WO2018008740A1 - Method for judging onset possibility of large bowel cancer - Google Patents

Method for judging onset possibility of large bowel cancer Download PDF

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WO2018008740A1
WO2018008740A1 PCT/JP2017/024956 JP2017024956W WO2018008740A1 WO 2018008740 A1 WO2018008740 A1 WO 2018008740A1 JP 2017024956 W JP2017024956 W JP 2017024956W WO 2018008740 A1 WO2018008740 A1 WO 2018008740A1
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Prior art keywords
methylation
cpg sites
colorectal cancer
methylation rate
ulcerative colitis
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PCT/JP2017/024956
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French (fr)
Japanese (ja)
Inventor
楠　正人
裕二 問山
光司 田中
荒木　俊光
彰 三井
竹鼻　健司
努 梅澤
Original Assignee
有限会社ハヌマット
Ｅａファーマ株式会社
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Priority claimed from PCT/JP2016/070330 external-priority patent/WO2018008153A1/en
Application filed by 有限会社ハヌマット, Ｅａファーマ株式会社 filed Critical 有限会社ハヌマット
Priority to EP21152410.3A priority Critical patent/EP3845669A1/en
Priority to CN201780041963.8A priority patent/CN109415771A/en
Priority to EP17824343.2A priority patent/EP3483282A4/en
Priority to EP21211939.0A priority patent/EP4023162A2/en
Priority to US16/315,961 priority patent/US20190241970A1/en
Priority to KR1020197000171A priority patent/KR20190045146A/en
Priority to JP2018526453A priority patent/JP7094881B2/en
Publication of WO2018008740A1 publication Critical patent/WO2018008740A1/en
Priority to US17/500,346 priority patent/US20220022851A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
    • A61B1/303—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor for the vagina, i.e. vaginoscopes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
    • A61B1/31—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor for the rectum, e.g. proctoscopes, sigmoidoscopes, colonoscopes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
    • A61B1/32—Devices for opening or enlarging the visual field, e.g. of a tube of the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/02—Instruments for taking cell samples or for biopsy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/02—Instruments for taking cell samples or for biopsy
    • A61B10/06—Biopsy forceps, e.g. with cup-shaped jaws
    • C—CHEMISTRY; METALLURGY
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/26—Inoculator or sampler
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/02—Instruments for taking cell samples or for biopsy
    • A61B2010/0216—Sampling brushes
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    • C12Q2523/00—Reactions characterised by treatment of reaction samples
    • C12Q2523/10—Characterised by chemical treatment
    • C12Q2523/125—Bisulfite(s)
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/165—Mathematical modelling, e.g. logarithm, ratio
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis, and a kit for collecting a rectal mucosa specimen used in the method.
  • This application claims priority based on PCT / JP2016 / 70330 filed on July 8, 2016 and Japanese Patent Application No. 2017-007725 filed in Japan on January 19, 2017, the contents of which are incorporated herein by reference. Incorporate.
  • Ulcerative colitis is an inflammatory bowel disease whose cause is unknown, which mainly causes ulcers and erosions in the colonic mucosa. Complete cure is very difficult, and remission and relapse are repeated. Symptoms include local symptoms of the large intestine such as diarrhea, abdominal pain and mucous stool, and systemic symptoms such as fever, vomiting, tachycardia and anemia. Patients with ulcerative colitis are more likely to develop colorectal cancer. Therefore, early detection and treatment of colorectal cancer is important in patients with ulcerative colitis.
  • Patent Document 1 discloses methylation of five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c in neoplastic tissues in patients with ulcerative colitis.
  • the methylation rate is significantly higher than that of the non-neoplastic ulcerative colitis tissue. Therefore, the methylation rate of the five miRNA genes in the biological sample collected from the colonic mucosa, which is a non-cancerous part, is It can be used as a marker for the development of colorectal cancer in patients with ulcerative colitis.
  • the present invention relates to a method for determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis by a method that is less invasive than endoscopy and less burdensome on the patient, and a rectum provided for the method
  • An object is to provide a kit for collecting a mucosal specimen.
  • CpG sites cytosine-phosphodiester bond-guanine
  • DMR 112 differently methylated regions
  • the present invention provides the following methods [1] to [34] for determining the possibility of developing colon cancer, a DNA methylation rate analysis marker, and a colonic mucosa collection kit.
  • a method for determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis One DNA present in each methylation variable region represented by methylation variable region numbers 1-112 shown in Tables 1 to 4 in DNA collected from a biological sample collected from a patient with human ulcerative colitis
  • a determination step of determining the likelihood of developing colon cancer in a patient with colitis The average methylation rate of the methylation variable region is an average value of the methylation rates of all CpG sites whose methylation rates were measured in the measurement
  • the multivariate discriminant measures the methylation rate of one or more CpG sites present in the methylation variable region including the average methylation rate as a variable
  • a discriminant value that is a value of the multivariate discriminant based on the average methylation rate of the methylation variable region calculated based on the methylation rate measured in the measurement step and the multivariate discriminant
  • the discriminant value is greater than or equal to a preset reference discriminant value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the multivariate discriminant includes, as a variable, an average methylation rate of two or more methylation variable regions selected from the methylation variable regions represented by methylation variable region numbers 1-112. [3] The method for determining the likelihood of developing colon cancer. [5] The multivariate discriminant includes, as a variable, an average methylation rate of three or more methylation variable regions selected from the methylation variable regions represented by methylation variable region numbers 1-112. [3] The method for determining the likelihood of developing colon cancer. [6] The multivariate discriminant is defined by using an average methylation rate of one or more methylation variable regions selected from the group consisting of methylation variable regions represented by methylation variable region numbers 1 to 58 as a variable.
  • the multivariate discriminant is defined by using an average methylation rate of one or more methylation variable regions selected from the group consisting of methylation variable regions represented by methylation variable region numbers 1 to 11 as a variable.
  • a method for determining the likelihood of developing colorectal cancer in a patient with human ulcerative colitis One or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 in DNA collected from biological samples collected from human ulcerative colitis patients
  • a measurement process for measuring the methylation rate Determination step of determining the possibility of developing colon cancer in the human ulcerative colitis patient based on the methylation rate measured in the measurement step and a preset reference value or a preset multivariate discriminant
  • the reference value is a value for discriminating between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient, each set for the methylation rate of each CpG site
  • the multivariate discriminant includes, as a variable, the methylation rate of at least one CpG site among the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80.
  • a method for determining the likelihood of developing colorectal cancer [9] The method for determining the likelihood of developing colon cancer according to the above [8], wherein in the measurement step, the methylation rate of 2 to 10 CpG sites is measured. [10] In the determination step, represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, 31, 45, 64, 65, 67, 77, 79, and 80 One or more of the CpG sites in the nucleotide sequence to be methylated has a methylation rate equal to or lower than a preset reference value, or SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, When one or more of the CpG sites in the base sequences represented by 32-44, 46-63, 66, 68-76, and 78 have a methylation rate equal to or higher than a preset reference value, The method for determining the likelihood of developing colon cancer according to [8] or [9] above, wherein it is determined that the human ulcerative colitis patient is likely
  • the methylation rate of the CpG site in the base sequence represented by SEQ ID NOs: 1 to 32 is measured
  • the determination step one or more of the CpG sites in the base sequence represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, and 31 are methyl Of the CpG site in the base sequence represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32
  • the above [8] to [10] wherein when the methylation rate is equal to or higher than a preset reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • a method for determining the likelihood of developing colorectal cancer is determining the likelihood of developing colorectal cancer.
  • methylation rate among CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14-18, 21-24, 26, 27, 29, and 31 Is the number of CpG sites whose pre-set reference value is less than or equal to a preset reference value, and methylation among CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32
  • the sum of the number of CpG sites with a rate equal to or higher than a preset reference value is 3 or more, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the methylation rate of the CpG site in the base sequences represented by SEQ ID NOs: 1 to 16 is measured,
  • the determination step at least one of the CpG sites in the base sequence represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 16 has a methylation rate equal to or lower than a preset reference value.
  • the human ulcerative colitis patient when one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10 and 13 have a methylation rate equal to or higher than a preset reference value, the human ulcerative colitis patient
  • the methylation rate of the CpG site in the base sequences represented by SEQ ID NOs: 1 to 9 is measured
  • the determination step one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 1 and 2 have a methylation rate equal to or lower than a preset reference value, or SEQ ID NOs: 3 to 9
  • the method for determining the likelihood of developing colorectal cancer according to any one of the above [8] to [10], wherein the determination is high.
  • the human ulcerative colitis patient is colon cancer
  • the method for determining the likelihood of developing colorectal cancer according to any one of [8] to [10] and [15], wherein it is determined that there is a high possibility of having developed.
  • the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66 is measured
  • the determination step one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65 have a methylation rate equal to or lower than a preset reference value, or SEQ ID NO: 33
  • the method for determining the likelihood of developing colorectal cancer according to any one of the above [8] to [10], wherein it is determined that there is a high possibility that the patient has colorectal cancer.
  • the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, and 67-80 is determined.
  • at least one of the CpG sites in the base sequences represented by SEQ ID NOs: 67, 77, 79, and 80 has a methylation rate equal to or lower than a preset reference value, or a sequence
  • one or more of the CpG sites in the base sequences represented by the numbers 33, 35, 36, 43, 68 to 76, and 78 have a methylation rate that is equal to or higher than a preset reference value.
  • the multivariate discriminant includes, as a variable, the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66,
  • the methylation rate of the CpG site in which the multivariate discriminant includes the methylation rate as a variable is measured
  • a discriminant value that is a value of the multivariate discriminant is calculated based on the methylation rate measured in the measurement step and the multivariate discriminant, and the discriminant value is set in advance as a reference discriminant value.
  • the method for determining the likelihood of developing colon cancer according to [8] or [9] above, wherein it is determined that the human ulcerative colitis patient is likely to develop colon cancer.
  • the multivariate discriminant is Including rate as a variable
  • the multivariate discriminant measures the methylation rate of the CpG site including the methylation rate as a variable
  • a discriminant value that is a value of the multivariate discriminant is calculated based on the methylation rate measured in the measurement step and the multivariate discriminant, and the discriminant value is set in advance as a reference discriminant value.
  • the multivariate discriminant is a logistic regression equation, a linear discriminant, an equation created by a naive Bayes classifier, or an equation created by a support vector machine A method for determining the likelihood of developing colorectal cancer.
  • the rectal mucosa tissue is A collection tool and a collection aid;
  • the sampling tool is A plate-like first clamping piece in which a first clamping surface for clamping the colonic mucosa is formed at one end;
  • a plate-like second clamping piece in which a second clamping surface for clamping the colonic mucosa is formed at one end;
  • a connecting portion that connects the first clamping piece and the second clamping piece in a state of being opposed to each other at an end portion where the first clamping surface and the second clamping surface are not formed;
  • Have At least one of the first clamping surface and the second clamping surface is cup-shaped
  • the collection aid is A frustoconical sampling tool introduction part having a slit on the side wall;
  • a rod-shaped gripping part Have One end of the gripping part is connected to the vicinity of the edge part of the larger outer diameter of the sampling tool introduction part, The slit is provided from the edge of the smaller outer diameter of the sampling tool introduction part toward the edge of the larger outer
  • a method for determining the likelihood of developing cancer [28]
  • the sampling tool is Having a first bent portion on the end side where the first clamping surface is formed with respect to the center of the first clamping piece;
  • the method for determining the likelihood of developing colorectal cancer according to [27] further including a second bent portion on an end side where the second clamping surface is formed with respect to a center portion of the second clamping piece.
  • a colonic mucosa collection kit comprising a collection tool and a collection aid.
  • the sampling tool is A plate-like first clamping piece in which a first clamping surface for clamping the colonic mucosa is formed at one end; A plate-like second clamping piece in which a second clamping surface for clamping the colonic mucosa is formed at one end; A connecting portion that connects the first clamping piece and the second clamping piece in a state of being opposed to each other at an end portion where the first clamping surface and the second clamping surface are not formed; Have At least one of the first clamping surface and the second clamping surface is cup-shaped,
  • the collection aid is A frustoconical sampling tool introduction part having a slit on the side wall; A rod-shaped gripping part; Have One end of the gripping part is connected to the vicinity of the edge part of the larger outer diameter of the sampling tool introduction part, The slit is provided from the edge of the smaller outer diameter of the sampling tool introduction part toward the edge of the larger outer diameter, The width of the slit is wider than the width of one end of the first clamping piece and the one
  • the sampling tool is Having a first bent portion on the end side where the first clamping surface is formed with respect to the center of the first clamping piece;
  • the collection aid has a through hole in the rotation axis direction, the outer diameter of the larger one of the collection tool introduction part is 30 to 70 mm, and the length in the rotation axis direction is 50 to 150 mm,
  • a DNA fragment comprising a DNA fragment having a partial base sequence containing one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80, and comprising a patient with ulcerative colitis
  • a marker for DNA methylation rate analysis used for determining the possibility of developing colorectal cancer.
  • the methylation rate of a specific CpG site or the average methylation rate of a specific DMR in a genomic DNA is examined for a biological sample collected from a patient with ulcerative colitis.
  • the rectal mucosa collection kit according to the present invention enables the rectal mucosa to be collected relatively safely and simply from the patient's anus.
  • FIG. 1 is an explanatory diagram of a sampling tool 2A which is an embodiment of the sampling tool and a sampling tool 2B which is a modified example thereof.
  • FIG. 2 is an explanatory diagram of a sampling tool 2C which is a modification of the sampling tool 2A.
  • FIG. 3 is an explanatory diagram of a collection assisting tool 11 ⁇ / b> A that is one embodiment of the collection assisting tool 11.
  • FIG. 4 is an explanatory diagram of a collection assistance tool 11B that is a modification of the collection assistance tool 11A.
  • FIG. 5 is an explanatory diagram of a usage mode of the rectal mucosa collection kit.
  • FIG. 1 is an explanatory diagram of a sampling tool 2A which is an embodiment of the sampling tool and a sampling tool 2B which is a modified example thereof.
  • FIG. 2 is an explanatory diagram of a sampling tool 2C which is a modification of the sampling tool 2A.
  • FIG. 3 is an explanatory diagram of a collection assisting tool
  • FIG. 6A shows the result of cluster analysis based on the methylation level of CpG sites in the 32CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6B shows the result of principal component analysis based on the methylation level of CpG sites in the 32 CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6C shows the result of cluster analysis based on the methylation level of CpG sites in the 16 CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6D shows the result of principal component analysis based on the methylation level of CpG sites in the 16 CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6A shows the result of cluster analysis based on the methylation level of CpG sites in the 32CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6C shows the result of cluster analysis based on the methylation
  • FIG. 6E shows the result of cluster analysis based on the methylation level of CpG sites in the 9CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6F is the result of principal component analysis based on the methylation level of CpG sites in the 9CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 7A shows that in Example 1, among the CpG sites in the five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c, the absolute value of DiffScore is 30. It is the result of the cluster analysis based on the methylation level of 27 CpG sites which were super.
  • FIG. 7A shows that in Example 1, among the CpG sites in the five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c, the absolute value of DiffScore is 30. It is the result of the cluster analysis
  • Example 7B shows that in Example 1, among the CpG sites in the five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c, the absolute value of DiffScore is 30. It is the result of the principal component analysis based on the methylation level of 27 CpG sites which were super.
  • FIG. 8A is the result of cluster analysis based on the methylation level of CpG sites in the 34 CpG set selected as a result of comprehensive DNA methylation analysis in Example 2.
  • FIG. 8B shows the result of principal component analysis based on the methylation level of CpG sites in the 34 CpG set selected as a result of comprehensive DNA methylation analysis in Example 2.
  • FIG. 8A is the result of cluster analysis based on the methylation level of CpG sites in the 34 CpG set selected as a result of comprehensive DNA methylation analysis in Example 2.
  • FIG. 8B shows the result of principal component analysis based on the methylation level of CpG
  • FIG. 9 shows the CpG site (cg10931190) in the base sequence represented by SEQ ID NO: 34, the CpG site (cg136767149) in the base sequence represented by SEQ ID NO: 37, and SEQ ID NO: 56 in Example 2.
  • FIG. 3 is an ROC curve of a test for the presence or absence of colon cancer in a patient with ulcerative colitis, using as a marker the methylation rate of three CpG sites in the CpG site (cg14516100) in the base sequence.
  • FIG. 10A shows the result of cluster analysis based on the methylation level of CpG sites in the 18 CpG set selected as a result of comprehensive DNA methylation analysis in Example 3.
  • FIG. 10A shows the result of cluster analysis based on the methylation level of CpG sites in the 18 CpG set selected as a result of comprehensive DNA methylation analysis in Example 3.
  • FIG. 10B shows the result of principal component analysis based on the methylation level of CpG sites in the 18 CpG set selected as a result of comprehensive DNA methylation analysis in Example 3.
  • FIG. 11 shows the results of cluster analysis based on the methylation rates at DMR 112 sites (112 DMR sets) selected as a result of comprehensive DNA methylation analysis in Example 4.
  • FIG. 12 shows the result of principal component analysis based on the methylation rate of the 112DMR set selected as a result of comprehensive DNA methylation analysis in Example 4.
  • FIG. 13 shows, in Example 4, the average methylation rate of three DMRs, DMR represented by DMR number 2, DMR represented by DMR number 10, and DMR represented by DMR number 55, as a marker. It is a ROC curve of the test
  • the cytosine base of the CpG site in genomic DNA can undergo methylation modification at the 5th carbon.
  • the methylation rate of a CpG site refers to the amount of methylated cytosine base (methylated cytosine) and the methylation among CpG sites in a biological sample collected from an individual organism.
  • the amount of untreated cytosine base (unmethylated cytosine) is measured, and means the ratio (%) of the amount of methylated cytosine to the sum of both.
  • the average methylation rate of DMR is an arithmetic average value (arithmetic average value) or geometric average value (geometric average value) of methylation rates of a plurality of CpG sites present in DMR.
  • an average value other than this may be used.
  • the method for determining the likelihood of developing colorectal cancer according to the present invention is a method for determining the likelihood of developing colorectal cancer in human ulcerative colitis patients.
  • the methylation rate of ulcerative colitis patients who have not developed colorectal cancer (non-cancer ulcerative colitis patients) group and ulcerative colitis patients who have developed colorectal cancer (Carcinogenic ulcerative colitis patient) What is different from the group is used as a marker.
  • methylation rate of CpG sites or the average methylation rate of DMR serving as these markers is determined whether or not a human ulcerative colitis patient has developed colorectal cancer.
  • Visual discrimination is very difficult by using the methylation rate of a specific CpG site or the average methylation rate of a specific DMR as a marker used to determine the likelihood of developing colorectal cancer in patients with ulcerative colitis.
  • Early colorectal cancer in patients with ulcerative colitis can be detected more objectively and sensitively, and early detection can be expected.
  • the determination of the possibility of developing colon cancer in a patient with human ulcerative colitis based on the methylation rate of the CpG site as a marker may be performed based on the measured value of the methylation rate of the CpG site itself. You may perform based on the discriminant value calculated
  • the determination of the possibility of developing colon cancer in human ulcerative colitis patients based on the average methylation rate of DMR as a marker is based on the average methylation rate of DMR calculated from the methylation rate of two or more CpG sites in DMR. It may be performed based on the value itself, or may be performed based on a discriminant value obtained from this multivariate discriminant using a multivariate discriminant including the average methylation rate of DMR as a marker as a variable.
  • the CpG site and DMR used as markers in the present invention those having a methylation rate greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group are preferable.
  • the larger the difference between the two groups the more reliably the presence or absence of colorectal cancer can be detected.
  • the CpG site and DMR, which are markers in the present invention have a significantly higher methylation rate in patients with carcinogenic ulcerative colitis than in non-cancer ulcerative colitis patients, that is, the methylation rate becomes higher due to the onset of colon cancer.
  • the methylation rate of patients with carcinogenic ulcerative colitis may be significantly lower than that of non-cancer ulcerative colitis patients, that is, the methylation rate may be lowered due to the onset of colorectal cancer. .
  • CpG site and DMR used as markers in the present invention those having a small difference in methylation rate between a non-cancerous part and a cancerous part of the large intestine in the same carcinogenic ulcerative colitis patient are more preferable.
  • a methylation rate of CpG sites or an average methylation rate of DMR as an index, even when a biological sample collected from a non-cancerous site of a carcinogenic ulcerative colitis patient is used, cancer As in the case of using a biological sample collected from a site, it is possible to determine whether or not colon cancer has developed with high sensitivity.
  • the mucous membrane in the deep part of the large intestine must be collected using an endoscope or the like, and the burden on the patient is large, but the rectal mucosa near the anus can be collected relatively easily.
  • the rectal mucosa near the anus is used as a biological sample regardless of the position where the cancerous site is formed. Can be detected without omission.
  • the method of determining based on the measured value of the methylation rate of the CpG site itself specifically determines the possibility of developing colorectal cancer in human ulcerative colitis patients.
  • the CpG site used as a marker in the present invention is specifically one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80. Each base sequence is shown in Tables 5-12. In the base sequences in the table, CG in parentheses is a CpG site detected by the comprehensive DNA methylation analysis shown in Examples 1 to 3. A DNA fragment having a base sequence containing these CpG sites can be used as a DNA methylation rate analysis marker for determining the possibility of developing colon cancer in patients with ulcerative colitis.
  • the 32 CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 1 to 32 were used in the comprehensive DNA methylation analysis in Example 1 described later.
  • the methylation rate is greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group.
  • those with carcinogenic ulcerative colitis patients whose methylation rate is considerably lower than those of non-cancer ulcerative colitis are SEQ ID NOs: 1, 2, 11, 12, 14-18, 21-24, 26, 27, CpG sites in the base sequences represented by 29 and 31 (in the table, “ ⁇ ”), and the methylation rate of carcinogenic ulcerative colitis patients is significantly higher than that of non-cancer ulcerative colitis patients,
  • the CpG site used as a marker is not limited to these 32 CpG sites, and includes other CpG sites in the nucleotide sequences represented by SEQ ID NOs: 1-32.
  • 34 CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 33 to 66 were used in the comprehensive DNA methylation analysis in Example 2 described later.
  • the methylation rate is greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group.
  • the CpG sites in the nucleotide sequences represented by SEQ ID NOs: 45, 64, and 65 are those in which the methylation rate of carcinogenic ulcerative colitis patients is considerably lower than that of non-cancer ulcerative colitis patients (Table Among the nucleotide sequences represented by SEQ ID NOs: 33 to 44, 46 to 63, and 66, the methylation rate of carcinogenic ulcerative colitis patients is considerably higher than that of non-cancer ulcerative colitis patients. It is a CpG site in the middle ("+" in the table).
  • the CpG sites used as markers are not limited to these 34 CpG sites, and other CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66 are also included.
  • a reference value for distinguishing between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient is set in advance. Measured in the case of CpG sites marked as “+” in Tables 5-7 and CpG sites marked as “+” in Tables 8-12 out of 34 CpG sets and 18 CpG sets in 32 CpG sets. When the methylation rate is equal to or higher than a preset reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the reference value of each CpG site is experimentally determined as a threshold value that can measure the methylation rate of the CpG site in the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group, and distinguish both groups. be able to.
  • the standard value for methylation of an arbitrary CpG site is obtained by a general statistical method. Examples thereof are shown below, but the method of determining the reference value in the present invention is not limited to these.
  • the reference value for example, for any CpG site, patients who have not been diagnosed with colon cancer by pathological examination using biopsy tissue in endoscopic examination among patients with ulcerative colitis (non- DNA methylation of rectal mucosa of patients with cancer ulcerative colitis is measured. After measuring about several patients, the numerical value which represents the methylation of these patient groups by the average value or the median value, etc. can be calculated, and this can be made into a reference value.
  • DNA methylation of the rectal mucosa was measured respectively, and the mean or median etc.
  • a threshold value that distinguishes both values in consideration of the variation can be obtained and used as a reference value.
  • the CpG site used as a marker in the present invention only the CpG site in the base sequence represented by SEQ ID NOs: 1 to 16 may be used. These 16 CpG sites (hereinafter, collectively referred to as “16CpG set”) are, in the 32CpG set, the methylation rate between the non-cancerous site and the cancerous site of the colon of carcinogenic ulcerative colitis patients. The difference is small.
  • the CpG site used as a marker in the present invention it is also preferable to use only the CpG site in the nucleotide sequence represented by SEQ ID NOs: 1 to 9.
  • 9CpG set These nine CpG sites (hereinafter, collectively referred to as “9CpG set”) are, of the 16CpG set, the methylation rate between the non-cancerous site and the cancerous site of the colon of carcinogenic ulcerative colitis patients. The difference is smaller.
  • the determination step it is represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, 31, 45, 64, 65, 67, 77, 79, and 80.
  • One or more of the CpG sites in the base sequence have a methylation rate not higher than a preset reference value, or SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, 32 to
  • the sum of the number of CpG sites with a methylation rate equal to or higher than a preset reference value among CpG sites in the base sequences represented by ⁇ 44, 46-63, 66, 68-76 and 78 is 2 or more In the case of preferably 3 or more, more preferably 5 or more, it is possible to make a more accurate determination by determining that the human ulcerative colitis patient has a high possibility of developing colon cancer. .
  • the 32CpG set is used as a marker in the present invention, that is, when the methylation rate of the 32CpG set is measured in the measurement step, in the determination step, SEQ ID NOs: 1, 2, 11, 12, 14 to One or more of the CpG sites in the base sequences represented by 18, 21 to 24, 26, 27, 29, and 31 have a methylation rate equal to or lower than a preset reference value, or SEQ ID NO: When one or more CpG sites in the base sequence represented by 3 to 10, 13, 19, 20, 25, 28, 30, and 32 have a methylation rate equal to or higher than a preset reference value Furthermore, it is determined that the human ulcerative colitis patient has a high possibility of developing colorectal cancer.
  • methylation is performed among CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, and 31.
  • the number of CpG sites whose rate is less than or equal to a preset reference value, and methyl of the CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32 When the sum of the number of CpG sites with a conversion rate equal to or higher than a preset reference value is 3 or more, preferably 5 or more, the human ulcerative colitis patient may develop colon cancer By determining that the value is high, the determination can be performed with higher accuracy.
  • one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65 have a methylation rate.
  • the methylation rate is preset for at least one of the CpG sites in the base sequence represented by SEQ ID NOs: 33-44, 46-63, and 66, which are below the preset reference value. If it is equal to or higher than the reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the number of CpG sites having a methylation rate equal to or lower than a preset reference value among the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and the sequence Among the CpG sites in the base sequences represented by the numbers 33 to 44, 46 to 63, and 66, the sum with the number of CpG sites whose methylation rate is not less than a preset reference value is preferably 2 or more, preferably 3 As described above, when it is more preferably 5 or more, it can be determined with higher accuracy by determining that the human ulcerative colitis patient has a high possibility of developing colorectal cancer.
  • one or more CpG sites in the base sequence represented by SEQ ID NOs: 67, 77, 79, and 80 are methylated.
  • the rate is below a preset reference value, or one or more of the CpG sites in the base sequence represented by SEQ ID NOs: 33, 35, 36, 43, 68 to 76, and 78 are methylated.
  • the rate is equal to or higher than a preset reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the determination method according to the present invention among the CpG sites in the base sequences represented by SEQ ID NOs: 67, 77, 79, and 80, the number of CpG sites having a methylation rate equal to or lower than a preset reference value And the sum of the number of CpG sites having a methylation rate equal to or higher than a preset reference value among the CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, 68 to 76, and 78.
  • it is 2 or more, preferably 3 or more, more preferably 5 or more, it is determined that the human ulcerative colitis patient has a high possibility of developing colorectal cancer, thereby making a more accurate determination Can do.
  • one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 can be used as markers.
  • the CpG sites used as markers in the present invention are all 80 CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 1 to 80 (hereinafter sometimes collectively referred to as “80 CpG set”). May be the 32CpG set, the 16CpG set, the 9CpG set, the 34CpG set, or the 18CpG set.
  • the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group have small dispersion of the methylation rate.
  • the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group are excellent in that they have a high discrimination ability.
  • the 34CpG set and the 18CpG set are slightly less specific than the CpG site of the 32CpG set, the 16CpG set, and the CpG site of the 9CpG set, the sensitivity is very high. It is very suitable for primary screening test for ulcerative colitis.
  • the method of determining based on the value of the average methylation rate of a specific DMR is specifically a method of determining the likelihood of developing colorectal cancer in a human ulcerative colitis patient
  • the methylation rate of one or more CpG sites present in a specific DMR used as a marker in the present invention in DNA collected from a biological sample collected from a patient with human ulcerative colitis is measured.
  • the average methylation rate of the DMR calculated based on the methylation rate measured in the measurement step, and a reference value set in advance for the average methylation rate of each DMR.
  • a determination step of determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis is calculated as the average value of the methylation rates of all CpG sites whose methylation rates were measured in the measurement step among the CpG sites in the DMR.
  • the DMR used as a marker in the present invention is specifically one or more DMRs selected from the group consisting of DMRs represented by DMR numbers 1-112.
  • the position of each DMR on the chromosome and the corresponding gene are shown in Tables 13-16.
  • the base positions of the start point and end point of DMR in the table are based on the human genome sequence data set “GRCh37 / hg19”.
  • DMRs represented by DMR numbers 1-112 have a plurality of CpG site methylation rates in patients with non-cancer ulcerative colitis.
  • Group and carcinogenic ulcerative colitis patient group are greatly different.
  • the average methylation rate of DMR average value of methylation rates of a plurality of CpG sites present in DMR
  • carcinogenic ulcerative colitis patients is considerably lower than that of non-cancer ulcerative colitis patients.
  • DMR numbers 1, 3 to 20, 23 to 28, 31 to 46, 49 to 60, 62, 65 to 69, 71, 73, 74, 79, 81, 82, 84, 86, 87, 90 to 92, 95 , 101, 103, 109, 110, and 112 ("-" in the table)
  • the fairly high ones are DMR numbers 2, 21, 22, 29, 30, 47, 48, 61, 63, 64, 70, 72, 75-78, 80, 83, 85, 88, 89, 93, 94, 96 ⁇ 100, 102, 104-108, and 11
  • the average methylation rate of DMR when used as a marker, one of the DMRs represented by DMR numbers 1-112 may be used as a marker, and the DMR represented by DMR numbers 1-112 Any two or more locations selected from the group consisting of may be used as markers, and all of the DMRs represented by DMR numbers 1-112 may be used as markers.
  • the number of DMRs used as markers in the group consisting of DMRs represented by DMR numbers 1-112 is preferably 2 or more, and more than 3 More preferably, it is more preferably 4 or more, and still more preferably 5 or more.
  • the DMR whose methylation rate is used as a marker in the present invention is a DMR represented by DMR numbers 1 to 58 (hereinafter sometimes referred to as “58 DMR set”). 1 or more selected from the group consisting of, more preferably 2 or more selected from the 58 DMR set, more preferably 3 or more selected from the 58 DMR set. 4 or more selected from the 58 DMR set, and more preferably 5 or more selected from the 58 DMR set.
  • the number is one or more selected from the group consisting of DMRs represented by DMR numbers 1 to 11 (hereinafter sometimes collectively referred to as “11 DMR sets”), and selected from 11 DMR sets. 2 or more, more preferably 3 or more selected from the 11 DMR set, even more preferably 4 or more selected from the 11 DMR set, from the 11 DMR set. It is particularly preferable that the number is 5 or more selected.
  • the average methylation rate of each DMR may be an average value of the methylation rates of all CpG sites included in each DMR, and at least one CpG site is selected from all CpG sites included in each DMR. It is good also as selecting arbitrarily and making it the average value of the methylation rate of this selected CpG site.
  • the methylation rate of each CpG site can be measured in the same manner as the measurement of the methylation rate of the CpG site in the base sequence represented by SEQ ID NO: 1 or the like.
  • a reference value for distinguishing between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient is set in advance.
  • the average methylation rate of the measured DMR is equal to or higher than a preset reference value, It is determined that the patient with inflammation is likely to develop colorectal cancer.
  • human ulcerative colitis when the average methylation rate of the measured DMR is not more than a preset reference value It is determined that the patient is likely to have colorectal cancer.
  • the standard value of the average methylation rate of each DMR is a threshold value that can be used to measure the average methylation rate of the DMR of the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group, and to distinguish the two groups. It can be determined experimentally. Specifically, the standard value of the average methylation rate of DMR is obtained by a general statistical method.
  • the determination method includes the determination step including a methylation rate measured in the measurement step and a preset multivariate. Based on the discriminant, the possibility of developing colorectal cancer in the human ulcerative colitis patient can be determined.
  • the multivariate discriminant includes, as a variable, the methylation rate of one or more CpG sites among the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80.
  • the determination method includes the determination step including the methylation measured in the measurement step. Based on the average methylation rate of DMR calculated based on the conversion rate and a multivariate discriminant set in advance, the possibility of the onset of colon cancer in the human ulcerative colitis patient can be determined.
  • the multivariate discriminant includes, as a variable, the methylation rate of one or more CpG sites among the CpG sites in the 112DMR set.
  • the multivariate discriminant used in the present invention can be obtained by a general method used to discriminate between the two groups.
  • Examples of the multivariate discriminant include, but are not limited to, a logistic regression equation, a linear discriminant, a formula created with a naive Bayes classifier, or a formula created with a support vector machine. .
  • These multivariate discriminants include, for example, one of the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 for the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group or The methylation rate of two or more CpG sites can be measured, and the resulting methylation rate can be used as a variable to prepare a standard method.
  • the average methylation rate of one or more DMRs among the DMRs in the 112 DMR set was measured and obtained.
  • the methylation rate can be used as a variable, and can be prepared by a conventional method.
  • a reference discriminating value for discriminating between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient is set in advance.
  • the discriminant value which is the value of the multivariate discriminant used for the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group, is obtained. It can be experimentally determined as a threshold value for distinguishing both groups by comparing the discriminant values of the cancer ulcerative colitis patient group.
  • the methylation rate of the CpG site or the average methylation rate of the DMR in which the multivariate discriminant used includes the methylation rate as a variable.
  • a discriminant value which is a value of the multivariate discriminant is calculated based on the methylation rate measured in the measurement step and the multivariate discriminant, and the discriminant value is set in advance.
  • the reference discriminant value it is determined whether a human ulcerative colitis patient whose CpG site methylation rate or DMR average methylation rate has been measured has developed colorectal cancer.
  • the discriminant value is greater than or equal to a preset reference discriminant value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the multivariate discriminant used in the present invention is preferably a formula containing as a variable the methylation rate of one or more CpG sites selected from the group consisting of the 34 CpG sites, and selected from the group consisting of the 34 CpG sites. More preferably, the formula includes only the methylation rate of one or more CpG sites as a variable, and only the methylation rate of 2 to 10 CpG sites arbitrarily selected from the group consisting of the 34 CpG sites is used as a variable. More preferably, the formula includes only the methylation rate of 2 to 5 CpG sites arbitrarily selected from the group consisting of the 34 CpG sites as a variable.
  • the multivariate discriminant used in the present invention is preferably a formula containing as a variable the methylation rate of one or more CpG sites selected from the group consisting of the 18CpG sites, and selected from the group consisting of the 18CpG sites. More preferably, the formula includes only the methylation rate of one or more CpG sites as a variable, and only the methylation rate of 2 to 10 CpG sites arbitrarily selected from the group consisting of the 18 CpG sites is used as a variable. More preferably, the formula includes only the methylation rate of 2 to 5 CpG sites arbitrarily selected from the group consisting of the 18 CpG sites as a variable.
  • the CpG sites constituting the 34 CpG set and the 18 CpG set are arbitrarily selected from 2 to 10, preferably 2 to 5 CpG sites from these sets, and even when only the selected CpG sites are used. With sufficient sensitivity and specificity, it is possible to determine the possibility of developing colon cancer in human ulcerative colitis patients.
  • the CpG site in the base sequence represented by SEQ ID NO: 34 the CpG site in the base sequence represented by SEQ ID NO: 37, and the sequence represented by SEQ ID NO: 56
  • the likelihood of developing colon cancer can be determined with a sensitivity of about 96% and a specificity of about 92%.
  • labor and cost may be excessive.
  • CpG site As a marker from the CpG sites constituting the 34CpG set and the 18CpG set, it is an appropriate number of CpG sites that can be measured in clinical examinations, and is accurate to human ulcerative colon. The possibility of developing colorectal cancer in patients with inflammation can be determined.
  • the multivariate discriminant used in the present invention is preferably an expression including, as a variable, an average methylation rate of one or more DMRs selected from the group consisting of the 112 DMR set, and selected from the group consisting of the 112 DMR set. More preferably, the formula includes only the average methylation rate of two or more DMRs as a variable, and includes only the average methylation rate of three or more DMRs arbitrarily selected from the group consisting of the 112 DMR sets as a variable.
  • the formula includes only the average methylation rate of four or more DMRs arbitrarily selected from the group consisting of the 112 DMR set as a variable, and more preferably the formula is arbitrarily selected from the group consisting of the 112 DMR set.
  • a formula including only the average methylation rate of 5 or more DMRs as a variable is particularly preferable.
  • a formula including an average methylation rate of one or more DMRs selected from the group consisting of the 58 DMR sets as a variable is preferable, and an average methyl of two or more DMRs selected from the group consisting of the 58 DMR sets is preferable.
  • the formula includes only the conversion rate as a variable, and more preferably includes only the average methylation rate of 2 to 10 DMRs arbitrarily selected from the group consisting of the 58 DMR set as a variable. More preferably, the formula includes only the average methylation rate of 3 to 10 DMRs arbitrarily selected from the group consisting of the variables, and 5 to 10 DMRs arbitrarily selected from the group consisting of the 58 DMR sets. Particularly preferred is a formula containing only the average methylation rate of as a variable.
  • a formula including, as a variable, an average methylation rate of one or more DMRs selected from the group consisting of the 11 DMR sets is preferable, and two or more DMRs selected from the group consisting of the 11 DMR sets are used.
  • a formula including only the average methylation rate as a variable is more preferable, and a formula including only the average methylation rate of 2 to 10 DMRs arbitrarily selected from the group consisting of the 11 DMR sets as a variable is more preferable.
  • the formula includes only the average methylation rate of 3 to 10 DMRs arbitrarily selected from the group consisting of the set as a variable, and 5 to 10 sites arbitrarily selected from the group consisting of the 11 DMR sets. Particularly preferred is a formula containing only the average methylation rate of DMR as a variable.
  • the biological sample used in the determination method according to the present invention is a biological sample collected from a patient with human ulcerative colitis and is not particularly limited as long as it contains the genomic DNA of the patient.
  • blood, plasma, serum, tears, saliva, or the like may be used, or tissue pieces collected from other tissues such as the digestive tract mucosa or the liver may be used.
  • the biological sample used in the determination method according to the present invention is preferably the large intestine mucosa because it more strongly reflects the state of the large intestine, and can be collected in the rectal mucosa because it can be collected with relatively low invasiveness. More preferably.
  • the rectal mucosa of the large intestine can be easily collected using, for example, a kit for collecting large intestine mucosa described later.
  • the biological sample may be in a state where DNA can be extracted, and may be subjected to various pretreatments.
  • it may be a formalin fixed paraffin embedded (FFPE) tissue.
  • FFPE formalin fixed paraffin embedded
  • Extraction of DNA from a biological sample can be performed by a conventional method, and various commercially available DNA extraction / purification kits can also be used.
  • the method for measuring the methylation rate of a CpG site is not particularly limited as long as it is a method capable of distinguishing and quantifying a methylated cytosine base and an unmethylated cytosine base for a specific CpG site.
  • the methylation rate of the CpG site can be measured by appropriately modifying methods known in the art as they are or as necessary. Examples of methods for measuring the methylation rate of CpG sites include bisulfite sequencing, COBRA (Combined Bisulfite Restoration Analysis), and qAMP (quantitative analysis of DNA methylation using real-time PCR). In addition, you may perform using the MIAM (Microarray-based Integrated Analysis of Methylation by Isoschizomers) method.
  • the kit for collecting large intestine mucosa comprises a collection tool for collecting with the rectal mucosa sandwiched therein, and a collection aid for expanding the anus and allowing the collection tool to reach the surface of the large intestine mucosa from the anus.
  • the colonic mucosa collection kit according to the present invention will be described below with reference to FIGS.
  • FIG. 1 (A) to FIG. 1 (C) are explanatory views of a sampling tool 2A, which is an embodiment of the sampling tool 2 of the colonic mucosa sampling kit 1.
  • FIG. FIG. 1 (A) is a perspective view of a state in which the first holding piece 3a and the second holding piece 3b of the sampling tool 2A are not applied
  • FIG. 1 (B) is a perspective view of the applied state.
  • FIG. 1C is a partially enlarged view of the distal end portion having the clamping surface of the sampling tool 2A.
  • the sampling tool 2A includes a first clamping piece 3a, a second clamping piece 3b, a connecting portion 4, a first clamping surface 5a, and a second clamping surface 5b. And having.
  • the first clamping piece 3a is a plate-like member in which a first clamping surface 5a for clamping the colonic mucosa is formed at one end, and the second clamping piece 3b is a large intestine at one end. It is a plate-like member on which a second clamping surface 5b that clamps the mucous membrane is formed.
  • the first sandwiching piece 3a and the second sandwiching piece 3b are connected to each other at the end where the first sandwiching surface 5a and the second sandwiching surface 5b are not formed in the connecting portion 4 in a state of facing each other. Yes.
  • the shape of the first sandwiching piece 3a and the second sandwiching piece 3b may be plate-like or rod-like, and may be formed to have a certain length for collecting the rectal mucosa. Don't stick to the shape.
  • the length of the first clamping piece 3a and the second clamping piece 3b is preferably 50 to 250 mm, more preferably 100 to 200 mm, still more preferably 70 to 200 mm, and even more preferably 70 to 150 mm. Since the first clamping piece 3a and the second clamping piece 3b have a length within the above range, the colonic mucosa is easily clamped and collected from the anus.
  • At least one of the first clamping surface 5a and the second clamping surface 5b is preferably cup-shaped. Since at least one of them is a cup shape, when the edge 6a of the first clamping surface 5a and the edge 6b of the second clamping surface 5b are in contact with each other, a space is formed inside. Of the large intestine mucosa sandwiched between the first sandwiching surface 5a and the second sandwiching surface 5b, the portion accommodated in the space does not take much load when the large intestine mucosa is torn, thus destroying the tissue. Can be suppressed. As shown in FIG. 1, since both are cup-shaped, it is easier to collect the mucosa of the large intestine, and tissue destruction can be suppressed.
  • the inner diameters of the marginal portion 6a and the marginal portion 6b may be set to such a size that a necessary amount of large intestine mucosa can be collected.
  • the large intestine mucosa used in the determination method according to the present invention it is sufficient if a small amount of mucosa can be collected.
  • the inner diameters of the edge portion 6a and the edge portion 6b to 1 to 5 mm, preferably 2 to 3 mm, a sufficient amount of the large intestine mucosa can be collected without excessively damaging the large intestine mucosa.
  • the edge portion 6a and the edge portion 6b may be flat as long as they can be brought into close contact with each other, but are preferably serrated as shown in FIG.
  • the large intestine mucosa can be cut and collected with a relatively weak force by being sandwiched between the edge 6a 'and the edge 6b'.
  • the protruding portion 8a may be formed on the inner side of one of the first holding piece 3a and the second holding piece 3b, and the cylindrical portion 9a may be formed on the other side so as to face each other.
  • the tip of the protrusion 8a fits into the tube 9a. Since the tip of the protrusion 8a is fitted into the tube portion 9a, it is possible to stably collect the large intestine mucosa without the edge 6a and the edge 6b being displaced when the collection tool 2 is separated from the large intestine mucosa. it can.
  • FIG. 1D is an explanatory diagram of a sampling tool 2B which is a modification of the sampling tool 2A. More specifically, the first clamping piece 3a and the second clamping piece 3b of the sampling tool 2B are applied with force.
  • the 1st clamping piece 3a may have the 1st bending part 7a in the edge part side in which the 1st clamping surface 5a is formed rather than the center part.
  • the 2nd clamping piece 3b may have the 2nd bending part 7b in the edge part side in which the 2nd clamping surface 5b is formed rather than the center part.
  • the first sandwiching piece 3a and the second sandwiching piece 3b are inclined while maintaining a state in which they are opposed to each other on the distal end side where the sandwiching surface is formed with respect to the center portion, thereby assisting collection. It becomes easy to penetrate the slit 13 of the tool 11 and contact the colonic mucosa.
  • the bending angle ⁇ 1 is preferably 10 to 50 °, more preferably 20 to 40 °, and even more preferably 25 to 35 °.
  • the length from the first bent portion 7a to the tip portion of the first clamping surface 5a and the length from the second bent portion 7b to the tip portion of the second holding surface 5b are 20 to 60 mm. 30 to 50 mm is more preferable.
  • the length from the bent portion to the distal end portion of the clamping surface is within the above range, it is easier to collect the mucous membrane in a state where it passes through the slit 13 of the collection assisting tool 11.
  • FIG. 2 (A) to 2 (E) are explanatory views of a sampling tool 2C, which is another modification of the sampling tool 2A.
  • FIG. 2 (A) is a front view showing a state in which the first clamping piece 3a and the second clamping piece 3b of the sampling tool 2 are not pressed
  • FIG. 2 (B) is a plan view of the sampling tool 2C.
  • FIG. 2C is an enlarged view of the protruding portion 8b of the sampling tool 2C
  • FIG. 2D is an opening edge of the cylindrical portion 9b where the locking claw of the protruding portion 8b is at the tip of the sampling tool 2C.
  • FIG. 2E is a plan view showing a state in which the first clamping surface 5a and the second clamping surface 5b are bonded to each other at the distal end portion of the sampling tool 2C.
  • the collection tool 2 When collecting mucosal tissue from the rectum of the subject, the collection tool 2 is more in a closed state than in a state in which the distance between the first holding piece 3a and the second holding piece 3b is open. It is easy to penetrate the slit 13. Therefore, as shown by the protrusion 8b in FIG. 2, the protrusion of the collection tool 2 may be a locking claw. There may be one or more locking claws of the protruding portion 8b, and any number may be used as long as it can be locked to the overhanging portion of the opening edge of the cylindrical portion 9b.
  • the cylindrical portion 9b for mating the protruding portion 8b is provided with a protruding portion on the opening edge radially inward, and the locking claw of the protruding portion 8b protrudes from the opening edge of the cylindrical portion 9b.
  • the height of the locking claw of the protrusion 8b is such that the tip of the first clamping surface 5a and the second clamping surface 5b are close to each other when locked to the overhanging portion of the tube portion 9b.
  • the first sandwiching surface 5a and the second sandwiching surface 5b are not bonded to each other, and the first sandwiching piece 3a and the second sandwiching piece 3b are further applied, whereby the tip of the protruding portion 8b.
  • first clamping surface 5a and the second clamping surface 5b can be bonded without penetrating through the bottom of the cylindrical portion 9b. This stabilizes the front end portions of the first clamping surface 5a and the second clamping surface 5b close to each other without applying force to the first clamping piece 3a and the second clamping piece 3b of the sampling tool 2. be able to.
  • the collection tool 2 penetrates the slit 13 of the collection assisting tool 11 in the state of FIG. 2D and the tip part contacts the rectal mucosa tissue, the first clamping piece 3a and the second clamping piece 3b are applied with force. Then, a part of the mucosal tissue is sandwiched and the state shown in FIG. 2E is obtained, and the mucosal tissue is collected.
  • the sampling tool 2 may be provided with a buffering portion 10a corresponding to a portion between the connecting portion and the bent portion in the first holding piece 3a and the second holding piece 3b, respectively.
  • the buffer portion 10a is provided with an elastic portion 10b at the tip thereof, and is bonded to each other by the elastic portion 10b in a state where the locking claw of the projection portion 8b is locked to the protruding portion of the opening edge portion of the cylindrical portion 9b ( FIG. 2 (D)).
  • the sampling tool 2 can maintain the state which the latching claw of the projection part 8b latched to the overhang
  • FIG. 3 (A) and 3 (B) are explanatory views of a collection assisting tool 11A which is an embodiment of the collection assisting tool 11.
  • FIG. FIG. 3A is a perspective view seen from the lower side of the collection assisting tool 11A
  • FIG. 3B is a bottom view seen from the slit side of the collection assisting tool 11A.
  • the collection assisting tool 11A has a collection tool introducing part 12, a slit 13, and a gripping part 14.
  • the sampling tool introduction part 12 is a truncated cone-shaped member having a slit 13 on the side wall.
  • the sampling tool introduction unit 12 is inserted into the anus from the distal edge 15 having a small outer diameter, and the sampling tool 2 is inserted from the proximal edge 16 having a large outer diameter.
  • the collection tool introduction part 12 may have a through hole in the rotation axis direction. From the viewpoint of ease of insertion into the anus, the outer diameter of the hand edge 16 is preferably 30 to 70 mm, and more preferably 40 to 50 mm.
  • the outer diameter of the distal edge portion 15 is preferably 10 to 30 mm, and more preferably 15 to 25 mm.
  • the length of the collection tool introduction part 12 in the rotation axis direction is preferably 50 to 150 mm, more preferably 70 to 130 mm, and even more preferably 80 to 120 mm.
  • the slit 13 is provided from the front edge portion 15 of the sampling tool introduction portion 12 toward the hand edge portion 16.
  • the presence of the slit 13 reaching the distal edge 15 in a part of the side wall of the collection tool introducing portion 12 increases the freedom of movement of the distal end portion of the collection tool 2 in the intestine, and in the rectum having a complicated internal structure, Large intestine mucosa can be collected more easily.
  • the slit 13 may be set at any position of the collection tool introduction unit 12.
  • the slit 13 is preferably on the side close to the gripping portion 14 as shown in FIG.
  • the number of the slits 13 provided in the collection tool introduction part 12 may be one, or two or more.
  • the width of the slit 13 is the same as that of the first clamping surface 5a of the collection tool 2 in a state where the edge 6a and the edge 6b are in contact with each other. It is designed wider than the width of the second clamping surface 5b. Further, the width of the slit 13 may be constant, but as shown in FIG. 3B, it is preferable that the slit 13 becomes wider from the front edge portion 15 toward the hand edge portion 16 side. For example, the width L 1 (see FIG.
  • the width L 2 (see FIG. 3B) of the slit 13 on the front edge portion 15 side is preferably 7 to 15 mm
  • the width L 3 of the slit 13 on the hand edge portion 16 side is preferably 10 to 20 mm. Note that two or more slits 13 may be formed on the wall surface of the collection tool introduction portion 12.
  • the length of the gripping portion 14 is preferably 50 to 150 mm, more preferably 70 to 130 mm, from the viewpoint of ease of gripping with a hand.
  • the shape of the gripping portion 14 may be any shape as long as it is easy to grip, and may be, for example, a plate shape, a rod shape, or other shapes.
  • FIG. 4 is an explanatory diagram of a collection assisting tool 11B which is a modification of the collection assisting tool 11A.
  • 4A is a perspective view seen from the upper side of the collection assisting tool 11B
  • FIG. 4B is a perspective view seen from the lower side.
  • 4 (C) to 4 (G) are a front view, a plan view, a bottom view, a left side view, and a right side view of the collection assisting tool 11B, respectively.
  • the gripping part of the collection assisting tool may be a hollow rod having an opening at the bottom and reinforced with a rib.
  • FIG. 5 is an explanatory diagram showing a mode of use of the colonic mucosa collection kit 1 according to the present invention.
  • the collection assisting tool 11 is inserted from the distal edge portion 15 into the anus of the subject from whom the colonic mucosa is collected.
  • the sampling tool 2 is introduced from the opening on the side of the hand edge 16 in a state where the grip 14 is held and stabilized with one hand.
  • the introduced collection tool 2 penetrates the slit 13 from the tip and reaches the surface of the large intestine mucosa.
  • the colonic mucosa can be collected by pulling out the sampling tool 2 from the slit 13 while the colonic mucosa is sandwiched between the clamping surface 5a and the clamping surface 5b of the sampling tool 2 (the clamping surface 5).
  • Example 1 Among the patients with ulcerative colitis, 8 patients (7 men, 1 woman) who were diagnosed with colon cancer by pathological diagnosis by biopsy tissue in endoscopy and who underwent surgery (7 men, 1 woman) , DNA in colonic mucosa collected from 8 patients (7 men, 1 woman) who have undergone surgery other than cancer (non-cancer UC patients) who are refractory to medical treatment ulcerative colitis In contrast, the methylation rate of the CpG site was comprehensively analyzed.
  • the average age of 8 UC cancer patients was 47.1 ⁇ 12.4 years, and the average disease duration was 11.4 ⁇ 7.3 years.
  • the average age of the 8 non-cancer UC patients was 44.3 ⁇ 16.4 years, and the average disease duration was 6.5 ⁇ 5.2 years.
  • Hybridization Hybridization buffer was added to the precipitated DNA, and the mixture was reacted with Hybridization Oven (manufactured by Illumina) at 48 ° C. for 1 hour to dissolve the DNA.
  • the dissolved DNA was incubated with a 95 ° C.
  • Microsample Incubator manufactured by SciGene
  • the reaction was carried out for 16 hours or more in Hybridization Oven at 48 ° C., and the probe on the BeadChip and the single-stranded DNA were hybridized.
  • [ ⁇ value] [Methylated fluorescence intensity] ⁇ ([Methylated fluorescence intensity] + [Unmethylated fluorescence intensity] +100)
  • GenomeStudio and software Methylation Module (Version: 1.9.0) were used for DNA methylation quantification and DNA methylation level comparison analysis.
  • the setting conditions of GenomeStudio are as follows.
  • the cancer patient samples were narrowed down to those with little fluctuation in DNA methylation level. That is, the unbiased variance var of the ⁇ value of 24 samples of UC cancer patients (3 sites ⁇ 8 samples of each site) was obtained, and 16 CpG sites having an unbiased variance var value smaller than 0.05 were selected.
  • the 16 CpG sites are collectively referred to as a “16 CpG set”.
  • Nine CpG sites with an unbiased variance var value smaller than 0.03 were further narrowed down from the 16 CpG set.
  • these nine CpG sites are collectively referred to as a “9CpG set”.
  • Table 17 shows the results of each CpG site of the 32CpG set.
  • CpG sites with # in the “16CpG” column indicate those included in the 16CpG set
  • CpG sites with # in the “9CpG” column indicate those included in the 9CpG set.
  • Example 2 In addition to the patients with ulcerative colitis of Example 1, 24 patients (UC cancer patients) who were diagnosed with colorectal cancer by pathological diagnosis with biopsy tissue by endoscopy and who underwent surgery, and medical treatment Comprehensive CpG site methylation rate for DNA in colon mucosa collected from 24 patients with refractory ulcerative colitis who have undergone surgery other than cancer (non-cancer UC patients) was analyzed.
  • the DNA used for the analysis of the methylation rate of the CpG site was extracted from the FFPE sample collected from the rectal mucosal tissue of a patient with ulcerative colitis in the same manner as in Example 1, and the whole genome was amplified. Quantification and comparative analysis of DNA methylation levels were performed, and DiffScore calculation, cluster analysis, and principal component analysis were performed using the results.
  • CpG biomarker candidates were extracted from comprehensive DNA methylation analysis data. Specifically, first, 324 CpG sites having an absolute value of ⁇ value exceeding 0.2 were extracted from 485,577 CpG sites.
  • FIG. 9 shows a ROC (Receiver-Operating-Characteristic) curve.
  • the AUC area under the ROC curve
  • Example 3 The DNA methylation level ( ⁇ value) of each CpG site of the specimen collected from the rectum of the ulcerative colitis patient obtained in Example 1 and the DNA methylation of each CpG site of the ulcerative colitis patient obtained in Example 2 CpG biomarker candidates were extracted from the conversion level ( ⁇ value).
  • Example 4 The average methylation rate of each DMR (average ⁇ value; methylation level of CpG sites present in each DMR) of specimens collected from the rectums of 24 UC cancer patients and 24 non-cancer UC patients obtained in Example 2 ( DMR biomarker candidates were extracted from the arithmetic average value of ⁇ value).
  • a logistic regression model was obtained using the numerical values ( ⁇ values) of the three DMR methylation levels of specimens collected from the rectums of 24 UC cancer patients and 24 non-cancer UC patients. Based on the discriminant, a UC cancer patient and a non-cancer UC patient were discriminated. As a result, the sensitivity (the ratio of patients evaluated as positive among UC cancer patients) was 95.8%, and the specificity (the ratio of patients evaluated as negative among non-cancer UC patients) was 95.8%. The positive predictive value (the proportion of UC cancer patients out of those evaluated as positive) is 95.8%, and the negative predictive value (the proportion of non-cancer UC patients out of patients evaluated as negative) is 95%.
  • FIG. 13 shows the ROC curve.
  • AUC area under the ROC curve

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Abstract

The present invention provides a method for judging the onset possibility of large bowel cancer in a human ulcerative colitis patient, etc. The method comprises a measurement step for measuring methylation ratios at one or more CpG sites existing in specific differentially methylated regions in DNA collected from a biological sample that is obtained from the human ulcerative colitis patient, and a judgment step for judging the onset possibility of large bowel cancer in the human ulcerative colitis patient on the basis of the average methylation ratio of the differentially methylated regions, said average methylation ratio being calculated from the measured methylation ratios, and preset standard values or a preset multivariable discriminant, wherein: the standard values are values having been set for the methylation ratios in the respective differentially methylated regions for discriminating a cancerous ulcerative colitis patient from a non-cancerous ulcerative colitis patient; and the multivariable discriminant contains, as variables, the average methylation ratios at one or more differentially methylated regions among the specific differentially methylated regions.

Description

大腸癌発症可能性の判定方法How to determine the likelihood of developing colorectal cancer
　本発明は、ヒト潰瘍性大腸炎患者の大腸癌発症の可能性を判定する方法、及び当該方法に供される直腸粘膜検体を採取するためのキットに関する。
　本願は、２０１６年７月８日に出願されたＰＣＴ／ＪＰ２０１６／７０３３０および２０１７年１月１９日に日本に出願された特願２０１７－００７７２５号に基づく優先権を主張し、その内容をここに援用する。 The present invention relates to a method for determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis, and a kit for collecting a rectal mucosa specimen used in the method.
This application claims priority based on PCT / JP2016 / 70330 filed on July 8, 2016 and Japanese Patent Application No. 2017-007725 filed in Japan on January 19, 2017, the contents of which are incorporated herein by reference. Incorporate.
　潰瘍性大腸炎は、主に大腸粘膜に潰瘍やびらんができる、原因不明の炎症性腸疾患である。完治は非常に困難であり、緩解・再燃を繰り返す。症状としては、下痢や腹痛、粘血便等の大腸の局所症状や、発熱、嘔吐、頻脈、貧血などの全身症状がある。潰瘍性大腸炎患者では大腸癌を発症しやすい。このため、潰瘍性大腸炎患者では、大腸癌の早期発見及び治療が重要である。 Ulcerative colitis is an inflammatory bowel disease whose cause is unknown, which mainly causes ulcers and erosions in the colonic mucosa. Complete cure is very difficult, and remission and relapse are repeated. Symptoms include local symptoms of the large intestine such as diarrhea, abdominal pain and mucous stool, and systemic symptoms such as fever, vomiting, tachycardia and anemia. Patients with ulcerative colitis are more likely to develop colorectal cancer. Therefore, early detection and treatment of colorectal cancer is important in patients with ulcerative colitis.
　一般的に大腸癌の早期発見のための検査は、通常、内視鏡検査により行われる。しかしながら、早期の大腸癌を視認により検出することは、手技者の技量によるところが大きく、一般的に困難である。特に潰瘍性大腸炎患者では、元々腸粘膜の炎症が酷いため、早期大腸癌の検出は非常に難しい。また、内視鏡検査は侵襲性が高く、患者の負担も大きいという問題もある。 Generally, examination for early detection of colorectal cancer is usually performed by endoscopy. However, it is generally difficult to visually detect early colorectal cancer because it depends largely on the skill of the operator. In particular, in patients with ulcerative colitis, detection of early colon cancer is very difficult because inflammation of the intestinal mucosa is originally severe. In addition, endoscopy has a problem that it is highly invasive and burdens the patient.
　一方で、特許文献１には、潰瘍性大腸炎患者において、腫瘍性組織におけるｍｉＲ－１、ｍｉＲ－９、ｍｉＲ－１２４、ｍｉＲ－１３７、及びｍｉＲ－３４ｂ／ｃの５種のｍｉＲＮＡ遺伝子のメチル化率は、非腫瘍性潰瘍性大腸炎組織に比べて有意に高いこと、よって、非癌部である結腸粘膜から採取された生体試料中の前記５種のｍｉＲＮＡ遺伝子のメチル化率は、潰瘍性大腸炎患者の大腸癌発症のマーカーとし得ることが報告されている。 On the other hand, Patent Document 1 discloses methylation of five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c in neoplastic tissues in patients with ulcerative colitis. The methylation rate is significantly higher than that of the non-neoplastic ulcerative colitis tissue. Therefore, the methylation rate of the five miRNA genes in the biological sample collected from the colonic mucosa, which is a non-cancerous part, is It can be used as a marker for the development of colorectal cancer in patients with ulcerative colitis.
国際公開第２０１４／１５１５５１号International Publication No. 2014/151551
　本発明は、ヒト潰瘍性大腸炎患者の大腸癌発症の可能性を、内視鏡検査よりも侵襲性が低く、より患者の負担が小さい方法で判定する方法、及び当該方法に供される直腸粘膜検体を採取するためのキットを提供することを目的とする。 The present invention relates to a method for determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis by a method that is less invasive than endoscopy and less burdensome on the patient, and a rectum provided for the method An object is to provide a kit for collecting a mucosal specimen.
　本発明者らは、上記課題を解決すべく鋭意検討した結果、潰瘍性大腸炎患者のゲノムＤＮＡ中のＣｐＧサイト（シトシン－ホスホジエステル結合－グアニン）のメチル化率を網羅的に調べたところ、大腸癌を発症した患者と大腸癌を発症していない患者においてメチル化率に顕著な差がある、８０種のＣｐＧサイトを見出し、また、別に１１２のメチル化可変領域（differentially methylated region、「ＤＭＲ」ということがある。）を見出し、本発明を完成させた。 As a result of intensive studies to solve the above problems, the present inventors have comprehensively investigated the methylation rate of CpG sites (cytosine-phosphodiester bond-guanine) in the genomic DNA of ulcerative colitis patients. We found 80 CpG sites with significant differences in the methylation rate between patients who developed colorectal cancer and those who did not develop colorectal cancer, and 112 differently methylated regions ("DMR"). ”) And the present invention was completed.
　すなわち、本発明は、以下の［１］～［３４］の大腸癌発症可能性の判定方法、ＤＮＡメチル化率分析用マーカー、及び大腸粘膜採取用キットを提供する。
［１］ヒト潰瘍性大腸炎患者の大腸癌発症可能性を判定する方法であって、
　ヒト潰瘍性大腸炎患者から採取された生体試料から回収されたＤＮＡ中の、表１～４に記載のメチル化可変領域番号１～１１２で表される各メチル化可変領域中に存在する１個以上のＣｐＧサイトのメチル化率を測定する測定工程と、
　前記測定工程において測定されたメチル化率に基づいて算出されたメチル化可変領域の平均メチル化率と、予め設定された基準値又は予め設定された多変量判別式に基づいて、前記ヒト潰瘍性大腸炎患者の大腸癌発症の可能性を判定する判定工程
を有し、
　前記メチル化可変領域の平均メチル化率が、当該メチル化可変領域中のＣｐＧサイトのうち、前記測定工程においてメチル化率が測定された全てのＣｐＧサイトのメチル化率の平均値であり、
　前記基準値が、各メチル化可変領域の平均メチル化率に対してそれぞれ設定された、発癌潰瘍性大腸炎患者と非癌潰瘍性大腸炎患者を識別するための値であり、
　前記多変量判別式が、前記メチル化可変領域番号１～１１２で表されるメチル化可変領域のうちの１か所以上のメチル化可変領域の平均メチル化率を変数として含む、
大腸癌発症可能性の判定方法。 That is, the present invention provides the following methods [1] to [34] for determining the possibility of developing colon cancer, a DNA methylation rate analysis marker, and a colonic mucosa collection kit.
[1] A method for determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis,
One DNA present in each methylation variable region represented by methylation variable region numbers 1-112 shown in Tables 1 to 4 in DNA collected from a biological sample collected from a patient with human ulcerative colitis A measuring step for measuring the methylation rate of the above CpG site;
Based on the average methylation rate of the methylation variable region calculated based on the methylation rate measured in the measurement step, and a preset reference value or a preset multivariate discriminant, the human ulcerative property A determination step of determining the likelihood of developing colon cancer in a patient with colitis,
The average methylation rate of the methylation variable region is an average value of the methylation rates of all CpG sites whose methylation rates were measured in the measurement step among the CpG sites in the methylation variable region,
The reference value is a value for discriminating between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient, each set for the average methylation rate of each methylation variable region,
The multivariate discriminant includes, as a variable, an average methylation rate of one or more methylation variable regions among the methylation variable regions represented by the methylation variable region numbers 1-112.
A method for determining the likelihood of developing colorectal cancer.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
［２］　前記判定工程において、メチル化可変領域番号１、３～２０、２３～２８、３１～４６、４９～６０、６２、６５～６９、７１、７３、７４、７９、８１、８２、８４、８６、８７、９０～９２、９５、１０１、１０３、１０９、１１０、及び１１２で表されるメチル化可変領域のうち１か所以上が、平均メチル化率が予め設定された基準値以下である、又は、メチル化可変領域番号２、２１、２２、２９、３０、４７、４８、６１，６３、６４、７０、７２、７５～７８、８０、８３、８５、８８、８９、９３、９４、９６～１００、１０２、１０４～１０８、及び１１１で表されるメチル化可変領域のうち１か所以上が、平均メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［１］の大腸癌発症可能性の判定方法。
［３］　前記測定工程において、前記多変量判別式がその平均メチル化率を変数として含むメチル化可変領域中に存在する１個以上のＣｐＧサイトのメチル化率を測定し、
　前記判定工程において、前記測定工程において測定されたメチル化率に基づいて算出されたメチル化可変領域の平均メチル化率と前記多変量判別式に基づいて当該多変量判別式の値である判別値を算出し、当該判別値が予め設定された基準判別値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［１］の大腸癌発症可能性の判定方法。
［４］　前記多変量判別式が、メチル化可変領域番号１～１１２で表されるメチル化可変領域から選択される２か所以上のメチル化可変領域の平均メチル化率を変数として含む、前記［３］の大腸癌発症可能性の判定方法。
［５］　前記多変量判別式が、メチル化可変領域番号１～１１２で表されるメチル化可変領域から選択される３か所以上のメチル化可変領域の平均メチル化率を変数として含む、前記［３］の大腸癌発症可能性の判定方法。
［６］　前記多変量判別式が、メチル化可変領域番号１～５８で表されるメチル化可変領域からなる群より選択される１か所以上のメチル化可変領域の平均メチル化率を変数として含む、前記［３］の大腸癌発症可能性の判定方法。
［７］　前記多変量判別式が、メチル化可変領域番号１～１１で表されるメチル化可変領域からなる群より選択される１か所以上のメチル化可変領域の平均メチル化率を変数として含む、前記［３］の大腸癌発症可能性の判定方法。 [2] In the determination step, methylation variable region numbers 1, 3 to 20, 23 to 28, 31 to 46, 49 to 60, 62, 65 to 69, 71, 73, 74, 79, 81, 82, 84 , 86, 87, 90 to 92, 95, 101, 103, 109, 110, and 112, at least one of the methylation variable regions has an average methylation rate that is equal to or lower than a preset reference value. Yes or methylation variable region number 2, 21, 22, 29, 30, 47, 48, 61, 63, 64, 70, 72, 75 to 78, 80, 83, 85, 88, 89, 93, 94 , 96 to 100, 102, 104 to 108, and 111, when at least one of the methylation variable regions has an average methylation rate equal to or higher than a preset reference value, the human ulcerative property A patient with colitis has developed colorectal cancer [1] The method for determining the likelihood of developing colorectal cancer according to [1].
[3] In the measurement step, the multivariate discriminant measures the methylation rate of one or more CpG sites present in the methylation variable region including the average methylation rate as a variable,
In the determination step, a discriminant value that is a value of the multivariate discriminant based on the average methylation rate of the methylation variable region calculated based on the methylation rate measured in the measurement step and the multivariate discriminant When the discriminant value is greater than or equal to a preset reference discriminant value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer. A method for determining the likelihood of developing cancer.
[4] The multivariate discriminant includes, as a variable, an average methylation rate of two or more methylation variable regions selected from the methylation variable regions represented by methylation variable region numbers 1-112. [3] The method for determining the likelihood of developing colon cancer.
[5] The multivariate discriminant includes, as a variable, an average methylation rate of three or more methylation variable regions selected from the methylation variable regions represented by methylation variable region numbers 1-112. [3] The method for determining the likelihood of developing colon cancer.
[6] The multivariate discriminant is defined by using an average methylation rate of one or more methylation variable regions selected from the group consisting of methylation variable regions represented by methylation variable region numbers 1 to 58 as a variable. [3] The method for determining the possibility of developing colorectal cancer according to [3].
[7] The multivariate discriminant is defined by using an average methylation rate of one or more methylation variable regions selected from the group consisting of methylation variable regions represented by methylation variable region numbers 1 to 11 as a variable. [3] The method for determining the possibility of developing colorectal cancer according to [3].
［８］　ヒト潰瘍性大腸炎患者の大腸癌発症可能性を判定する方法であって、
　ヒト潰瘍性大腸炎患者から採取された生体試料から回収されたＤＮＡ中の、配列番号１～８０で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を測定する測定工程と、
　前記測定工程において測定されたメチル化率と、予め設定された基準値又は予め設定された多変量判別式に基づいて、前記ヒト潰瘍性大腸炎患者の大腸癌発症の可能性を判定する判定工程
を有し、
　前記基準値が、各ＣｐＧサイトのメチル化率に対してそれぞれ設定された、発癌潰瘍性大腸炎患者と非癌潰瘍性大腸炎患者を識別するための値であり、
　前記多変量判別式が、前記配列番号１～８０で表される塩基配列中のＣｐＧサイトのうち少なくとも１か所のＣｐＧサイトのメチル化率を変数として含む、
大腸癌発症可能性の判定方法。
［９］前記測定工程において、２～１０個のＣｐＧサイトのメチル化率を測定する、前記［８］の大腸癌発症可能性の判定方法。
［１０］　前記判定工程において、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、３１、４５、６４、６５、６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３～１０、１３、１９、２０、２５、２８、３０、３２～４４、４６～６３、６６、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］又は［９］の大腸癌発症可能性の判定方法。
［１１］　前記測定工程において、配列番号１～３２で表される塩基配列中のＣｐＧサイトのメチル化率を測定し、
　前記判定工程において、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、及び３１で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３～１０、１３、１９、２０、２５、２８、３０、及び３２で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］～［１０］のいずれかの大腸癌発症可能性の判定方法。
［１２］　前記判定工程において、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、及び３１で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３～１０、１３、１９、２０、２５、２８、３０、及び３２で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が３以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］～［１１］のいずれかの大腸癌発症可能性の判定方法。
［１３］　前記測定工程において、配列番号１～１６で表される塩基配列中のＣｐＧサイトのメチル化率を測定し、
　前記判定工程において、配列番号１、２、１１、１２、１４～１６で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３～１０、１３で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］～［１０］のいずれかの大腸癌発症可能性の判定方法。
［１４］　前記判定工程において、配列番号１、２、１１、１２、１４～１６で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３～１０、１３で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が３以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］～［１０］及び［１３］のいずれかの大腸癌発症可能性の判定方法。
［１５］　前記測定工程において、配列番号１～９で表される塩基配列中のＣｐＧサイトのメチル化率を測定し、
　前記判定工程において、配列番号１及び２で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３～９で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］～［１０］のいずれかの大腸癌発症可能性の判定方法。
［１６］　前記判定工程において、配列番号１及び２で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３～９で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が３以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］～［１０］及び［１５］のいずれかの大腸癌発症可能性の判定方法。
［１７］　前記測定工程において、配列番号３３～６６で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を測定し、
　前記判定工程において、配列番号４５、６４、及び６５で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３３～４４、４６～６３、及び６６で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］～［１０］のいずれかの大腸癌発症可能性の判定方法。
［１８］　前記判定工程において、配列番号４５、６４、及び６５で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３３～４４、４６～６３、及び６６で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が２以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］～［１０］及び［１７］のいずれかの大腸癌発症可能性の判定方法。
［１９］　前記測定工程において、配列番号３３、３５、３６、４３、６７～８０で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を測定し、
　前記判定工程において、配列番号６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３３、３５、３６、４３、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］～［１０］のいずれかの大腸癌発症可能性の判定方法。
［２０］　前記判定工程において、配列番号６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３３、３５、３６、４３、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が２以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］～［１０］及び［１９］のいずれかの大腸癌発症可能性の判定方法。
［２１］　前記和が５以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［１２］、［１４］、［１６］、［１８］、又は［２０］の大腸癌発症可能性の判定方法。
［２２］　前記多変量判別式が、配列番号３３～６６で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を変数として含み、　前記測定工程において、前記多変量判別式がそのメチル化率を変数として含むＣｐＧサイトのメチル化率を測定し、
　前記判定工程において、前記測定工程において測定されたメチル化率と前記多変量判別式に基づいて当該多変量判別式の値である判別値を算出し、当該判別値が予め設定された基準判別値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］又は［９］の大腸癌発症可能性の判定方法。
［２３］　前記多変量判別式が、配列番号３３、３５、３６、４３、６７～８０で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を変数として含み、
　前記測定工程において、前記多変量判別式がそのメチル化率を変数として含むＣｐＧサイトのメチル化率を測定し、
　前記判定工程において、前記測定工程において測定されたメチル化率と前記多変量判別式に基づいて当該多変量判別式の値である判別値を算出し、当該判別値が予め設定された基準判別値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、前記［８］又は［９］の大腸癌発症可能性の判定方法。
［２４］　前記多変量判別式が、ロジスティック回帰式、線形判別式、ナイーブベイズ分類器で作成された式、又はサポートベクターマシンで作成された式である、前記［１］～［２３］のいずれかの大腸癌発症可能性の判定方法。
［２５］　前記生体試料が、腸管組織である、前記［１］～［２４］のいずれかの大腸癌発症可能性の判定方法。
［２６］　前記生体試料が、直腸粘膜組織である、前記［１］～［２５］のいずれかの大腸癌発症可能性の判定方法。 [8] A method for determining the likelihood of developing colorectal cancer in a patient with human ulcerative colitis,
One or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 in DNA collected from biological samples collected from human ulcerative colitis patients A measurement process for measuring the methylation rate;
Determination step of determining the possibility of developing colon cancer in the human ulcerative colitis patient based on the methylation rate measured in the measurement step and a preset reference value or a preset multivariate discriminant Have
The reference value is a value for discriminating between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient, each set for the methylation rate of each CpG site,
The multivariate discriminant includes, as a variable, the methylation rate of at least one CpG site among the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80.
A method for determining the likelihood of developing colorectal cancer.
[9] The method for determining the likelihood of developing colon cancer according to the above [8], wherein in the measurement step, the methylation rate of 2 to 10 CpG sites is measured.
[10] In the determination step, represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, 31, 45, 64, 65, 67, 77, 79, and 80 One or more of the CpG sites in the nucleotide sequence to be methylated has a methylation rate equal to or lower than a preset reference value, or SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, When one or more of the CpG sites in the base sequences represented by 32-44, 46-63, 66, 68-76, and 78 have a methylation rate equal to or higher than a preset reference value, The method for determining the likelihood of developing colon cancer according to [8] or [9] above, wherein it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
[11] In the measurement step, the methylation rate of the CpG site in the base sequence represented by SEQ ID NOs: 1 to 32 is measured,
In the determination step, one or more of the CpG sites in the base sequence represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, and 31 are methyl Of the CpG site in the base sequence represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32 The above [8] to [10], wherein when the methylation rate is equal to or higher than a preset reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer. A method for determining the likelihood of developing colorectal cancer.
[12] In the determination step, methylation rate among CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14-18, 21-24, 26, 27, 29, and 31 Is the number of CpG sites whose pre-set reference value is less than or equal to a preset reference value, and methylation among CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32 When the sum of the number of CpG sites with a rate equal to or higher than a preset reference value is 3 or more, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer, [8] The method for determining the likelihood of developing colorectal cancer according to any one of [11].
[13] In the measurement step, the methylation rate of the CpG site in the base sequences represented by SEQ ID NOs: 1 to 16 is measured,
In the determination step, at least one of the CpG sites in the base sequence represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 16 has a methylation rate equal to or lower than a preset reference value. Alternatively, when one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10 and 13 have a methylation rate equal to or higher than a preset reference value, the human ulcerative colitis patient The method for determining the likelihood of developing colorectal cancer according to any one of the above [8] to [10], wherein it is determined that there is a high possibility that the patient has colorectal cancer.
[14] Among the CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 16 in the determination step, CpG sites having a methylation rate equal to or lower than a preset reference value When the sum of the number and the number of CpG sites having a methylation rate equal to or higher than a preset reference value among the CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10 and 13 is 3 or more, The method for determining the likelihood of developing colorectal cancer according to any one of [8] to [10] and [13] above, wherein it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
[15] In the measurement step, the methylation rate of the CpG site in the base sequences represented by SEQ ID NOs: 1 to 9 is measured,
In the determination step, one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 1 and 2 have a methylation rate equal to or lower than a preset reference value, or SEQ ID NOs: 3 to 9 Possibility that the human ulcerative colitis patient develops colorectal cancer when one or more of the CpG sites in the represented base sequence has a methylation rate that is equal to or higher than a preset reference value The method for determining the likelihood of developing colorectal cancer according to any one of the above [8] to [10], wherein the determination is high.
[16] In the determination step, among the CpG sites in the base sequences represented by SEQ ID NOS: 1 and 2, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and SEQ ID NOS: 3 to 9 When the sum of the CpG sites in the base sequence represented by the number of CpG sites having a methylation rate equal to or higher than a preset reference value is 3 or more, the human ulcerative colitis patient is colon cancer The method for determining the likelihood of developing colorectal cancer according to any one of [8] to [10] and [15], wherein it is determined that there is a high possibility of having developed.
[17] In the measurement step, the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66 is measured,
In the determination step, one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65 have a methylation rate equal to or lower than a preset reference value, or SEQ ID NO: 33 A patient with human ulcerative colitis when one or more of the CpG sites in the base sequences represented by -44, 46-63, and 66 have a methylation rate equal to or higher than a preset reference value The method for determining the likelihood of developing colorectal cancer according to any one of the above [8] to [10], wherein it is determined that there is a high possibility that the patient has colorectal cancer.
[18] In the determination step, among the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and the SEQ ID NO: When the sum of the CpG sites in the base sequences represented by 33 to 44, 46 to 63 and 66 and the number of CpG sites having a methylation rate equal to or higher than a preset reference value is 2 or more, The method for determining the likelihood of developing colorectal cancer according to any one of [8] to [10] and [17], wherein the human ulcerative colitis patient is determined to have a high probability of developing colorectal cancer.
[19] In the measurement step, the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, and 67-80 is determined. Measure and
In the determination step, at least one of the CpG sites in the base sequences represented by SEQ ID NOs: 67, 77, 79, and 80 has a methylation rate equal to or lower than a preset reference value, or a sequence When one or more of the CpG sites in the base sequences represented by the numbers 33, 35, 36, 43, 68 to 76, and 78 have a methylation rate that is equal to or higher than a preset reference value, The method for determining the possibility of developing colorectal cancer according to any one of the above [8] to [10], wherein it is determined that a human ulcerative colitis patient is likely to develop colorectal cancer.
[20] In the determination step, among the CpG sites in the base sequences represented by SEQ ID NOs: 67, 77, 79, and 80, the number of CpG sites having a methylation rate equal to or lower than a preset reference value; The sum of the CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, 68 to 76, and 78 with a methylation rate equal to or higher than a preset reference value is 2 When the above is true, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer, and the possibility of developing colorectal cancer according to any of [8] to [10] and [19] Judgment method.
[21] When the sum is 5 or more, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer, [12], [14], [16], [16] 18] or [20] The method for determining the likelihood of developing colorectal cancer.
[22] The multivariate discriminant includes, as a variable, the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66, In the process, the methylation rate of the CpG site in which the multivariate discriminant includes the methylation rate as a variable is measured,
In the determination step, a discriminant value that is a value of the multivariate discriminant is calculated based on the methylation rate measured in the measurement step and the multivariate discriminant, and the discriminant value is set in advance as a reference discriminant value. In the case described above, the method for determining the likelihood of developing colon cancer according to [8] or [9] above, wherein it is determined that the human ulcerative colitis patient is likely to develop colon cancer.
[23] Methylation of one or more CpG sites selected from the group consisting of CpG sites in the base sequence represented by SEQ ID NOs: 33, 35, 36, 43, 67-80, wherein the multivariate discriminant is Including rate as a variable,
In the measurement step, the multivariate discriminant measures the methylation rate of the CpG site including the methylation rate as a variable,
In the determination step, a discriminant value that is a value of the multivariate discriminant is calculated based on the methylation rate measured in the measurement step and the multivariate discriminant, and the discriminant value is set in advance as a reference discriminant value. In the case described above, the method for determining the likelihood of developing colon cancer according to [8] or [9] above, wherein it is determined that the human ulcerative colitis patient is likely to develop colon cancer.
[24] Any of the above [1] to [23], wherein the multivariate discriminant is a logistic regression equation, a linear discriminant, an equation created by a naive Bayes classifier, or an equation created by a support vector machine A method for determining the likelihood of developing colorectal cancer.
[25] The method for determining the likelihood of developing colorectal cancer according to any one of [1] to [24], wherein the biological sample is intestinal tissue.
[26] The method for determining the likelihood of developing colorectal cancer according to any one of [1] to [25], wherein the biological sample is rectal mucosal tissue.
［２７］　前記直腸粘膜組織が、
　採取具と、採取補助具とを備え、
　前記採取具は、
　一方の端部に大腸粘膜を挟持する第１の挟持面が形成されている板状の第１の挟持片と、
　一方の端部に大腸粘膜を挟持する第２の挟持面が形成されている板状の第２の挟持片と、
　前記第１の挟持片と前記第２の挟持片を、互いに対向した状態で、前記第１の挟持面及び前記第２の挟持面が形成されていない端部において連結する連結部と、
を有し、
　前記第１の挟持面及び前記第２の挟持面の少なくとも一方がカップ形状であり、
　前記採取補助具は、
　側壁にスリットを有する円錐台形状の採取具導入部と、
　棒状の把持部と、
を有し、
　前記把持部の一端が、前記採取具導入部の外径が大きい方の辺縁部近傍に連結しており、
　前記スリットは、前記採取具導入部の外径が小さい方の辺縁部から外径が大きい方の辺縁部に向かって設けられており、
　前記スリットの幅が、前記第１の挟持片の一方の端部と前記第２の挟持片の一方の端部の幅よりも広く、
前記採取具導入部の大きい方の外径が３０～７０ｍｍであり、回転軸方向の長さが５０～１５０ｍｍである、大腸粘膜採取用キットによって採取されたものである、前記［２６］の大腸癌発症可能性の判定方法。
［２８］　前記採取具が、
　前記第１の挟持片の中心部よりも前記第１の挟持面が形成されている端部側に、第１の屈曲部を有し、
　前記第２の挟持片の中心部よりも前記第２の挟持面が形成されている端部側に、第２の屈曲部を有する、前記［２７］の大腸癌発症可能性の判定方法。
［２９］　採取具と、採取補助具とを備える大腸粘膜採取用キットであり、
　前記採取具は、
　一方の端部に大腸粘膜を挟持する第１の挟持面が形成されている板状の第１の挟持片と、
　一方の端部に大腸粘膜を挟持する第２の挟持面が形成されている板状の第２の挟持片と、
　前記第１の挟持片と前記第２の挟持片を、互いに対向した状態で、前記第１の挟持面及び前記第２の挟持面が形成されていない端部において連結する連結部と、
を有し、
　前記第１の挟持面及び前記第２の挟持面の少なくとも一方がカップ形状であり、
　前記採取補助具は、
　側壁にスリットを有する円錐台形状の採取具導入部と、
　棒状の把持部と、
を有し、
　前記把持部の一端が、前記採取具導入部の外径が大きい方の辺縁部近傍に連結しており、
　前記スリットは、前記採取具導入部の外径が小さい方の辺縁部から外径が大きい方の辺縁部に向かって設けられており、
　前記スリットの幅が、前記第１の挟持片の一方の端部と前記第２の挟持片の一方の端部の幅よりも広く、
　前記採取具導入部の大きい方の外径が３０～７０ｍｍであり、回転軸方向の長さが５０～１５０ｍｍである、大腸粘膜採取用キット。
［３０］　前記採取具が、
　前記第１の挟持片の中心部よりも前記第１の挟持面が形成されている端部側に、第１の屈曲部を有し、
　前記第２の挟持片の中心部よりも前記第２の挟持面が形成されている端部側に、第２の屈曲部を有する、前記［２９］の大腸粘膜採取用キット。
［３１］　前記第１の挟持面と前記第２の挟持面の両方がカップ形状である、前記［２９］又は［３０］の大腸粘膜採取用キット。
［３２］　前記採取補助具が、回転軸方向に貫通孔を有し、前記採取具導入部の大きい方の外径が３０～７０ｍｍ、回転軸方向の長さが５０～１５０ｍｍであり、
　前記カップ形状の辺縁部の内径が２～３ｍｍである、前記［２９］～［３１］のいずれかの大腸粘膜採取用キット。
［３３］　前記第１の挟持面と前記第２の挟持面の辺縁部が鋸歯状である、前記［２９］～［３２］のいずれかの大腸粘膜採取用キット。
［３４］　配列番号１～８０で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトを含む部分塩基配列を有するＤＮＡ断片からなり、潰瘍性大腸炎患者の大腸癌発症可能性を判定するために用いられる、ＤＮＡメチル化率分析用マーカー。 [27] The rectal mucosa tissue is
A collection tool and a collection aid;
The sampling tool is
A plate-like first clamping piece in which a first clamping surface for clamping the colonic mucosa is formed at one end;
A plate-like second clamping piece in which a second clamping surface for clamping the colonic mucosa is formed at one end;
A connecting portion that connects the first clamping piece and the second clamping piece in a state of being opposed to each other at an end portion where the first clamping surface and the second clamping surface are not formed;
Have
At least one of the first clamping surface and the second clamping surface is cup-shaped,
The collection aid is
A frustoconical sampling tool introduction part having a slit on the side wall;
A rod-shaped gripping part;
Have
One end of the gripping part is connected to the vicinity of the edge part of the larger outer diameter of the sampling tool introduction part,
The slit is provided from the edge of the smaller outer diameter of the sampling tool introduction part toward the edge of the larger outer diameter,
The width of the slit is wider than the width of one end of the first clamping piece and the one end of the second clamping piece,
The large intestine of the above [26], which is collected by a large intestine mucosa collection kit having a larger outer diameter of the collection tool introduction part of 30 to 70 mm and a length in the rotation axis direction of 50 to 150 mm. A method for determining the likelihood of developing cancer.
[28] The sampling tool is
Having a first bent portion on the end side where the first clamping surface is formed with respect to the center of the first clamping piece;
[27] The method for determining the likelihood of developing colorectal cancer according to [27], further including a second bent portion on an end side where the second clamping surface is formed with respect to a center portion of the second clamping piece.
[29] A colonic mucosa collection kit comprising a collection tool and a collection aid.
The sampling tool is
A plate-like first clamping piece in which a first clamping surface for clamping the colonic mucosa is formed at one end;
A plate-like second clamping piece in which a second clamping surface for clamping the colonic mucosa is formed at one end;
A connecting portion that connects the first clamping piece and the second clamping piece in a state of being opposed to each other at an end portion where the first clamping surface and the second clamping surface are not formed;
Have
At least one of the first clamping surface and the second clamping surface is cup-shaped,
The collection aid is
A frustoconical sampling tool introduction part having a slit on the side wall;
A rod-shaped gripping part;
Have
One end of the gripping part is connected to the vicinity of the edge part of the larger outer diameter of the sampling tool introduction part,
The slit is provided from the edge of the smaller outer diameter of the sampling tool introduction part toward the edge of the larger outer diameter,
The width of the slit is wider than the width of one end of the first clamping piece and the one end of the second clamping piece,
A large intestine mucosa collection kit having a larger outer diameter of the collection tool introduction part of 30 to 70 mm and a length in the rotation axis direction of 50 to 150 mm.
[30] The sampling tool is
Having a first bent portion on the end side where the first clamping surface is formed with respect to the center of the first clamping piece;
[29] The colonic mucosa collection kit according to [29], further including a second bent portion on an end portion side where the second clamping surface is formed with respect to a center portion of the second clamping piece.
[31] The kit for collecting large intestine mucosa according to [29] or [30], wherein both the first clamping surface and the second clamping surface are cup-shaped.
[32] The collection aid has a through hole in the rotation axis direction, the outer diameter of the larger one of the collection tool introduction part is 30 to 70 mm, and the length in the rotation axis direction is 50 to 150 mm,
The kit for collecting mucosa of large intestine according to any one of the above [29] to [31], wherein an inner diameter of the cup-shaped edge is 2 to 3 mm.
[33] The colonic mucosa collection kit according to any one of [29] to [32], wherein the edges of the first clamping surface and the second clamping surface are serrated.
[34] A DNA fragment comprising a DNA fragment having a partial base sequence containing one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80, and comprising a patient with ulcerative colitis A marker for DNA methylation rate analysis used for determining the possibility of developing colorectal cancer.
　本発明に係る大腸癌発症可能性の判定方法により、潰瘍性大腸炎患者から採取された生体試料について、ゲノムＤＮＡ中の特定のＣｐＧサイトのメチル化率又は特定のＤＭＲの平均メチル化率を調べることによって、大腸癌発症の可能性を判定することができる。　また、本発明に係る直腸粘膜採取用キットにより、患者の肛門から比較的安全かつ簡便に直腸粘膜を採取することができる。 According to the method for determining the likelihood of developing colon cancer according to the present invention, the methylation rate of a specific CpG site or the average methylation rate of a specific DMR in a genomic DNA is examined for a biological sample collected from a patient with ulcerative colitis. Thus, the possibility of developing colorectal cancer can be determined. Also, the rectal mucosa collection kit according to the present invention enables the rectal mucosa to be collected relatively safely and simply from the patient's anus.
図１は、採取具の一実施態様である採取具２Ａ及びその変形例である採取具２Ｂの説明図である。FIG. 1 is an explanatory diagram of a sampling tool 2A which is an embodiment of the sampling tool and a sampling tool 2B which is a modified example thereof. 図２は、採取具２Ａの変形例である採取具２Ｃの説明図である。FIG. 2 is an explanatory diagram of a sampling tool 2C which is a modification of the sampling tool 2A. 図３は、採取補助具１１の一実施態様である採取補助具１１Ａの説明図である。FIG. 3 is an explanatory diagram of a collection assisting tool 11 </ b> A that is one embodiment of the collection assisting tool 11. 図４は、採取補助具１１Ａの変形例である採取補助具１１Ｂの説明図である。FIG. 4 is an explanatory diagram of a collection assistance tool 11B that is a modification of the collection assistance tool 11A. 図５は直腸粘膜採取用キットの使用態様の説明図である。FIG. 5 is an explanatory diagram of a usage mode of the rectal mucosa collection kit. 図６Ａは、実施例１において、網羅的ＤＮＡメチル化解析の結果選抜された３２ＣｐＧセットのＣｐＧサイトのメチル化レベルに基づくクラスター解析の結果である。FIG. 6A shows the result of cluster analysis based on the methylation level of CpG sites in the 32CpG set selected as a result of comprehensive DNA methylation analysis in Example 1. 図６Ｂは、実施例１において、網羅的ＤＮＡメチル化解析の結果選抜された３２ＣｐＧセットのＣｐＧサイトのメチル化レベルに基づく主成分分析の結果である。FIG. 6B shows the result of principal component analysis based on the methylation level of CpG sites in the 32 CpG set selected as a result of comprehensive DNA methylation analysis in Example 1. 図６Ｃは、実施例１において、網羅的ＤＮＡメチル化解析の結果選抜された１６ＣｐＧセットのＣｐＧサイトのメチル化レベルに基づくクラスター解析の結果である。FIG. 6C shows the result of cluster analysis based on the methylation level of CpG sites in the 16 CpG set selected as a result of comprehensive DNA methylation analysis in Example 1. 図６Ｄは、実施例１において、網羅的ＤＮＡメチル化解析の結果選抜された１６ＣｐＧセットのＣｐＧサイトのメチル化レベルに基づく主成分分析の結果である。FIG. 6D shows the result of principal component analysis based on the methylation level of CpG sites in the 16 CpG set selected as a result of comprehensive DNA methylation analysis in Example 1. 図６Ｅは、実施例１において、網羅的ＤＮＡメチル化解析の結果選抜された９ＣｐＧセットのＣｐＧサイトのメチル化レベルに基づくクラスター解析の結果である。FIG. 6E shows the result of cluster analysis based on the methylation level of CpG sites in the 9CpG set selected as a result of comprehensive DNA methylation analysis in Example 1. 図６Ｆは、実施例１において、網羅的ＤＮＡメチル化解析の結果選抜された９ＣｐＧセットのＣｐＧサイトのメチル化レベルに基づく主成分分析の結果である。FIG. 6F is the result of principal component analysis based on the methylation level of CpG sites in the 9CpG set selected as a result of comprehensive DNA methylation analysis in Example 1. 図７Ａは、実施例１において、ｍｉＲ－１、ｍｉＲ－９、ｍｉＲ－１２４、ｍｉＲ－１３７、及びｍｉＲ－３４ｂ／ｃの５種のｍｉＲＮＡ遺伝子にあるＣｐＧサイトのうち、DiffScoreの絶対値が３０超であった２７個のＣｐＧサイトのメチル化レベルに基づくクラスター解析の結果である。FIG. 7A shows that in Example 1, among the CpG sites in the five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c, the absolute value of DiffScore is 30. It is the result of the cluster analysis based on the methylation level of 27 CpG sites which were super. 図７Ｂは、実施例１において、ｍｉＲ－１、ｍｉＲ－９、ｍｉＲ－１２４、ｍｉＲ－１３７、及びｍｉＲ－３４ｂ／ｃの５種のｍｉＲＮＡ遺伝子にあるＣｐＧサイトのうち、DiffScoreの絶対値が３０超であった２７個のＣｐＧサイトのメチル化レベルに基づく主成分分析の結果である。FIG. 7B shows that in Example 1, among the CpG sites in the five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c, the absolute value of DiffScore is 30. It is the result of the principal component analysis based on the methylation level of 27 CpG sites which were super. 図８Ａは、実施例２において、網羅的ＤＮＡメチル化解析の結果選抜された３４ＣｐＧセットのＣｐＧサイトのメチル化レベルに基づくクラスター解析の結果である。FIG. 8A is the result of cluster analysis based on the methylation level of CpG sites in the 34 CpG set selected as a result of comprehensive DNA methylation analysis in Example 2. 図８Ｂは、実施例２において、網羅的ＤＮＡメチル化解析の結果選抜された３４ＣｐＧセットのＣｐＧサイトのメチル化レベルに基づく主成分分析の結果である。FIG. 8B shows the result of principal component analysis based on the methylation level of CpG sites in the 34 CpG set selected as a result of comprehensive DNA methylation analysis in Example 2. 図９は、実施例２において、配列番号３４で表される塩基配列中のＣｐＧサイト（ｃｇ１０９３１１９０）、配列番号３７で表される塩基配列中のＣｐＧサイト（ｃｇ１３６７７１４９）、及び配列番号５６で表される塩基配列中のＣｐＧサイト（ｃｇ１４５１６１００）の３個のＣｐＧサイトのメチル化率をマーカーとした場合の、潰瘍性大腸炎患者の大腸癌発症の有無の検査のＲＯＣ曲線である。FIG. 9 shows the CpG site (cg10931190) in the base sequence represented by SEQ ID NO: 34, the CpG site (cg136767149) in the base sequence represented by SEQ ID NO: 37, and SEQ ID NO: 56 in Example 2. FIG. 3 is an ROC curve of a test for the presence or absence of colon cancer in a patient with ulcerative colitis, using as a marker the methylation rate of three CpG sites in the CpG site (cg14516100) in the base sequence. 図１０Ａは、実施例３において、網羅的ＤＮＡメチル化解析の結果選抜された１８ＣｐＧセットのＣｐＧサイトのメチル化レベルに基づくクラスター解析の結果である。FIG. 10A shows the result of cluster analysis based on the methylation level of CpG sites in the 18 CpG set selected as a result of comprehensive DNA methylation analysis in Example 3. 図１０Ｂは、実施例３において、網羅的ＤＮＡメチル化解析の結果選抜された１８ＣｐＧセットのＣｐＧサイトのメチル化レベルに基づく主成分分析の結果である。FIG. 10B shows the result of principal component analysis based on the methylation level of CpG sites in the 18 CpG set selected as a result of comprehensive DNA methylation analysis in Example 3. 図１１は、実施例４において、網羅的ＤＮＡメチル化解析の結果選抜されたＤＭＲ１１２か所（１１２ＤＭＲセット）のメチル化率に基づくクラスター解析の結果である。FIG. 11 shows the results of cluster analysis based on the methylation rates at DMR 112 sites (112 DMR sets) selected as a result of comprehensive DNA methylation analysis in Example 4. 図１２は、実施例４において、網羅的ＤＮＡメチル化解析の結果選抜された１１２ＤＭＲセットのメチル化率に基づく主成分分析の結果である。FIG. 12 shows the result of principal component analysis based on the methylation rate of the 112DMR set selected as a result of comprehensive DNA methylation analysis in Example 4. 図１３は、実施例４において、ＤＭＲ番号２で表されるＤＭＲ、ＤＭＲ番号１０で表されるＤＭＲ、及びＤＭＲ番号５５で表されるＤＭＲの３個のＤＭＲの平均メチル化率をマーカーとした場合の、潰瘍性大腸炎患者の大腸癌発症の有無の検査のＲＯＣ曲線である。FIG. 13 shows, in Example 4, the average methylation rate of three DMRs, DMR represented by DMR number 2, DMR represented by DMR number 10, and DMR represented by DMR number 55, as a marker. It is a ROC curve of the test | inspection of the presence or absence of colon cancer onset in the patient of ulcerative colitis.
＜大腸癌発症可能性の判定方法＞
　ゲノムＤＮＡ中のＣｐＧサイトのシトシン塩基は、５位の炭素がメチル化修飾を受け得る。本発明及び本願明細書において、ＣｐＧサイトのメチル化率とは、一の生物個体から採取された生体試料中のＣｐＧサイトのうち、メチル化されているシトシン塩基（メチル化シトシン）量とメチル化されていないシトシン塩基（非メチル化シトシン）量とを測定し、両者の和に対するメチル化シトシン量の割合（％）を意味する。また、本発明及び本願明細書において、ＤＭＲの平均メチル化率とは、ＤＭＲに存在する複数のＣｐＧサイトのメチル化率の相加平均値（算術平均値）又は相乗平均値（幾何平均値）を意味するが、これ以外の平均値でもよい。 <Method for determining the likelihood of developing colorectal cancer>
The cytosine base of the CpG site in genomic DNA can undergo methylation modification at the 5th carbon. In the present invention and the present specification, the methylation rate of a CpG site refers to the amount of methylated cytosine base (methylated cytosine) and the methylation among CpG sites in a biological sample collected from an individual organism. The amount of untreated cytosine base (unmethylated cytosine) is measured, and means the ratio (%) of the amount of methylated cytosine to the sum of both. In the present invention and the present specification, the average methylation rate of DMR is an arithmetic average value (arithmetic average value) or geometric average value (geometric average value) of methylation rates of a plurality of CpG sites present in DMR. However, an average value other than this may be used.
　本発明に係る大腸癌発症可能性の判定方法（以下、「本発明に係る判定方法」ということがある。）は、ヒト潰瘍性大腸炎患者の大腸癌発症可能性を判定する方法であって、ゲノムＤＮＡ中のＣｐＧサイト又はＤＭＲのうち、メチル化率が、大腸癌を発症していない潰瘍性大腸炎患者（非癌潰瘍性大腸炎患者）群と大腸癌を発症した潰瘍性大腸炎患者（発癌潰瘍性大腸炎患者）群とで差があるものをマーカーとすることを特徴とする。これらのマーカーとなるＣｐＧサイトのメチル化率又はＤＭＲの平均メチル化率を指標として、ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性の高低を判定する。特定のＣｐＧサイトのメチル化率又は特定のＤＭＲの平均メチル化率を潰瘍性大腸炎患者の大腸癌発症可能性を判定するために用いられるマーカーとすることにより、視認判別が非常に困難である潰瘍性大腸炎患者における早期大腸癌をより客観的かつ感度よく検出することができ、早期発見が期待できる。 The method for determining the likelihood of developing colorectal cancer according to the present invention (hereinafter sometimes referred to as the “method of determining the present invention”) is a method for determining the likelihood of developing colorectal cancer in human ulcerative colitis patients. Among the CpG sites or DMRs in genomic DNA, the methylation rate of ulcerative colitis patients who have not developed colorectal cancer (non-cancer ulcerative colitis patients) group and ulcerative colitis patients who have developed colorectal cancer (Carcinogenic ulcerative colitis patient) What is different from the group is used as a marker. Using the methylation rate of CpG sites or the average methylation rate of DMR serving as these markers as an index, it is determined whether or not a human ulcerative colitis patient has developed colorectal cancer. Visual discrimination is very difficult by using the methylation rate of a specific CpG site or the average methylation rate of a specific DMR as a marker used to determine the likelihood of developing colorectal cancer in patients with ulcerative colitis. Early colorectal cancer in patients with ulcerative colitis can be detected more objectively and sensitively, and early detection can be expected.
　マーカーとするＣｐＧサイトのメチル化率に基づくヒト潰瘍性大腸炎患者の大腸癌発症可能性の判定は、測定されたＣｐＧサイトのメチル化率の値自体に基づいて行ってもよく、マーカーとするＣｐＧサイトのメチル化率を変数として含む多変量判別式を用い、この多変量判別式から求められる判別値に基づいて行ってもよい。 The determination of the possibility of developing colon cancer in a patient with human ulcerative colitis based on the methylation rate of the CpG site as a marker may be performed based on the measured value of the methylation rate of the CpG site itself. You may perform based on the discriminant value calculated | required from this multivariate discriminant using the multivariate discriminant containing the methylation rate of a CpG site as a variable.
　マーカーとするＤＭＲの平均メチル化率に基づくヒト潰瘍性大腸炎患者の大腸癌発症可能性の判定は、ＤＭＲ中の２以上のＣｐＧサイトのメチル化率から算出されたＤＭＲの平均メチル化率の値自体に基づいて行ってもよく、マーカーとするＤＭＲの平均メチル化率を変数として含む多変量判別式を用い、この多変量判別式から求められる判別値に基づいて行ってもよい。 The determination of the possibility of developing colon cancer in human ulcerative colitis patients based on the average methylation rate of DMR as a marker is based on the average methylation rate of DMR calculated from the methylation rate of two or more CpG sites in DMR. It may be performed based on the value itself, or may be performed based on a discriminant value obtained from this multivariate discriminant using a multivariate discriminant including the average methylation rate of DMR as a marker as a variable.
　本発明においてマーカーとされるＣｐＧサイト及びＤＭＲとしては、メチル化率が非癌潰瘍性大腸炎患者群と発癌潰瘍性大腸炎患者群とで大きく相違するものが好ましい。両群の差が大きいほど、大腸癌の発症の有無をより確実に検出することができる。本発明においてマーカーとされるＣｐＧサイト及びＤＭＲは、発癌潰瘍性大腸炎患者のメチル化率が非癌潰瘍性大腸炎患者よりも有意に高い、すなわち、大腸癌の発症によりメチル化率が高くなるものであってもよく、発癌潰瘍性大腸炎患者のメチル化率が非癌潰瘍性大腸炎患者よりも有意に低い、すなわち、大腸癌の発症によりメチル化率が低くなるものであってもよい。 As the CpG site and DMR used as markers in the present invention, those having a methylation rate greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group are preferable. The larger the difference between the two groups, the more reliably the presence or absence of colorectal cancer can be detected. The CpG site and DMR, which are markers in the present invention, have a significantly higher methylation rate in patients with carcinogenic ulcerative colitis than in non-cancer ulcerative colitis patients, that is, the methylation rate becomes higher due to the onset of colon cancer. The methylation rate of patients with carcinogenic ulcerative colitis may be significantly lower than that of non-cancer ulcerative colitis patients, that is, the methylation rate may be lowered due to the onset of colorectal cancer. .
　本発明においてマーカーとされるＣｐＧサイト及びＤＭＲとしては、同一の発癌潰瘍性大腸炎患者において、大腸の非癌部位と癌部位とのメチル化率の差が小さいものがより好ましい。このようなＣｐＧサイトのメチル化率又はＤＭＲの平均メチル化率を指標とすることにより、発癌潰瘍性大腸炎患者の非癌部位から採取された生体試料が用いられた場合であっても、癌部位から採取された生体試料を用いた場合と同様に、高感度に大腸癌の発症の有無を判定することができる。例えば、大腸深部の粘膜は内視鏡等を用いて採取する必要があり、患者の負担が大きいが、肛門付近の直腸粘膜は比較的容易に採取できる。大腸の非癌部位と癌部位とのメチル化率の差が小さいＣｐＧサイト又はＤＭＲをマーカーとすることにより、癌部位が形成される位置にかかわらず、肛門付近の直腸粘膜を生体試料として大腸癌を発症した患者を漏れなく検出することができる。 As the CpG site and DMR used as markers in the present invention, those having a small difference in methylation rate between a non-cancerous part and a cancerous part of the large intestine in the same carcinogenic ulcerative colitis patient are more preferable. By using such a methylation rate of CpG sites or an average methylation rate of DMR as an index, even when a biological sample collected from a non-cancerous site of a carcinogenic ulcerative colitis patient is used, cancer As in the case of using a biological sample collected from a site, it is possible to determine whether or not colon cancer has developed with high sensitivity. For example, the mucous membrane in the deep part of the large intestine must be collected using an endoscope or the like, and the burden on the patient is large, but the rectal mucosa near the anus can be collected relatively easily. By using a CpG site or DMR with a small difference in methylation rate between a non-cancerous site and a cancerous site in the large intestine as a marker, the rectal mucosa near the anus is used as a biological sample regardless of the position where the cancerous site is formed. Can be detected without omission.
　本発明に係る判定方法のうち、測定されたＣｐＧサイトのメチル化率の値自体に基づいて判定を行う方法は、具体的には、ヒト潰瘍性大腸炎患者の大腸癌発症可能性を判定する方法であって、ヒト潰瘍性大腸炎患者から採取された生体試料から回収されたＤＮＡ中の、本発明においてマーカーとする特定の複数のＣｐＧサイトのメチル化率を測定する測定工程と、前記測定工程において測定されたメチル化率と、各ＣｐＧサイトに対して予めそれぞれ設定された基準値に基づいて、前記ヒト潰瘍性大腸炎患者の大腸癌発症の可能性を判定する判定工程を有する。 Among the determination methods according to the present invention, the method of determining based on the measured value of the methylation rate of the CpG site itself specifically determines the possibility of developing colorectal cancer in human ulcerative colitis patients. A method for measuring methylation rates of a plurality of specific CpG sites as markers in the present invention in DNA collected from a biological sample collected from a patient with human ulcerative colitis, and the measurement And a determination step of determining the possibility of developing colon cancer in the human ulcerative colitis patient based on the methylation rate measured in the step and a reference value set in advance for each CpG site.
　本発明においてマーカーとされるＣｐＧサイトは、具体的には、配列番号１～８０で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトである。各塩基配列を表５～１２に示す。表の塩基配列中、括弧内のＣＧは、実施例１～３で示す網羅的ＤＮＡメチル化解析で検出されるＣｐＧサイトである。これらのＣｐＧサイトを含む塩基配列を有するＤＮＡ断片は、潰瘍性大腸炎患者の大腸癌発症可能性を判定するためのＤＮＡメチル化率分析用マーカーとして用いることができる。 The CpG site used as a marker in the present invention is specifically one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80. Each base sequence is shown in Tables 5-12. In the base sequences in the table, CG in parentheses is a CpG site detected by the comprehensive DNA methylation analysis shown in Examples 1 to 3. A DNA fragment having a base sequence containing these CpG sites can be used as a DNA methylation rate analysis marker for determining the possibility of developing colon cancer in patients with ulcerative colitis.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
　配列番号１～３２で表される塩基配列中の括弧内の３２個のＣｐＧサイト（以下、まとめて「３２ＣｐＧセット」ということがある。）は、後記実施例１における網羅的ＤＮＡメチル化解析において、メチル化率が非癌潰瘍性大腸炎患者群と発癌潰瘍性大腸炎患者群とで大きく相違するものである。このうち、発癌潰瘍性大腸炎患者のメチル化率が非癌潰瘍性大腸炎患者よりもかなり低いものが、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、及び３１で表される塩基配列中のＣｐＧサイトであり（表中、「－」）、発癌潰瘍性大腸炎患者のメチル化率が非癌潰瘍性大腸炎患者よりもかなり高いものが、配列番号３～１０、１３、１９、２０、２５、２８、３０、及び３２で表される塩基配列中のＣｐＧサイトである（表中、「＋」）。マーカーとされるＣｐＧサイトは、これら３２個のＣｐＧサイトに限定されるものではなく、配列番号１～３２で表される塩基配列中の他のＣｐＧサイトも含まれる。 The 32 CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 1 to 32 (hereinafter sometimes collectively referred to as “32CpG sets”) were used in the comprehensive DNA methylation analysis in Example 1 described later. The methylation rate is greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group. Among them, those with carcinogenic ulcerative colitis patients whose methylation rate is considerably lower than those of non-cancer ulcerative colitis are SEQ ID NOs: 1, 2, 11, 12, 14-18, 21-24, 26, 27, CpG sites in the base sequences represented by 29 and 31 (in the table, “−”), and the methylation rate of carcinogenic ulcerative colitis patients is significantly higher than that of non-cancer ulcerative colitis patients, This is a CpG site in the base sequence represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32 (“+” in the table). The CpG site used as a marker is not limited to these 32 CpG sites, and includes other CpG sites in the nucleotide sequences represented by SEQ ID NOs: 1-32.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
　配列番号３３～６６で表される塩基配列中の括弧内の３４個のＣｐＧサイト（以下、まとめて「３４ＣｐＧセット」ということがある。）は、後記実施例２における網羅的ＤＮＡメチル化解析において、メチル化率が非癌潰瘍性大腸炎患者群と発癌潰瘍性大腸炎患者群とで大きく相違するものである。このうち、発癌潰瘍性大腸炎患者のメチル化率が非癌潰瘍性大腸炎患者よりもかなり低いものが、配列番号４５、６４、及び６５で表される塩基配列中のＣｐＧサイトであり（表中、「－」）、発癌潰瘍性大腸炎患者のメチル化率が非癌潰瘍性大腸炎患者よりもかなり高いものが、配列番号３３～４４、４６～６３、及び６６で表される塩基配列中のＣｐＧサイトである（表中、「＋」）。マーカーとされるＣｐＧサイトは、これら３４個のＣｐＧサイトに限定されるものではなく、配列番号３３～６６で表される塩基配列中の他のＣｐＧサイトも含まれる。 34 CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 33 to 66 (hereinafter, sometimes collectively referred to as “34 CpG set”) were used in the comprehensive DNA methylation analysis in Example 2 described later. The methylation rate is greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group. Among them, the CpG sites in the nucleotide sequences represented by SEQ ID NOs: 45, 64, and 65 are those in which the methylation rate of carcinogenic ulcerative colitis patients is considerably lower than that of non-cancer ulcerative colitis patients (Table Among the nucleotide sequences represented by SEQ ID NOs: 33 to 44, 46 to 63, and 66, the methylation rate of carcinogenic ulcerative colitis patients is considerably higher than that of non-cancer ulcerative colitis patients. It is a CpG site in the middle ("+" in the table). The CpG sites used as markers are not limited to these 34 CpG sites, and other CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66 are also included.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　配列番号３３、３５、３６、４３、６７～８０で表される塩基配列中の括弧内の１８個のＣｐＧサイト（以下、まとめて「１８ＣｐＧセット」ということがある。）は、後記実施例３における網羅的ＤＮＡメチル化解析において、メチル化率が非癌潰瘍性大腸炎患者群と発癌潰瘍性大腸炎患者群とで大きく相違するものである。このうち、発癌潰瘍性大腸炎患者のメチル化率が非癌潰瘍性大腸炎患者よりもかなり低いものが、配列番号６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトであり（表中、「－」）、発癌潰瘍性大腸炎患者のメチル化率が非癌潰瘍性大腸炎患者よりもかなり高いものが、配列番号３３、３５、３６、４３、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトである（表中、「＋」）。マーカーとされるＣｐＧサイトは、これら１８個のＣｐＧサイトに限定されるものではなく、配列番号３３、３５、３６、４３、６７～８０で表される塩基配列中の他のＣｐＧサイトも含まれる。 Eighteen CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, and 67 to 80 (hereinafter sometimes collectively referred to as “18 CpG set”) are described in Example 3 below. In the comprehensive DNA methylation analysis, the methylation rate is greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group. Among them, the CpG site in the base sequence represented by SEQ ID NOs: 67, 77, 79, and 80 has a methylation rate significantly lower in patients with carcinogenic ulcerative colitis than in non-cancer ulcerative colitis patients. ("-" In the table), those with carcinogenic ulcerative colitis patients whose methylation rate is considerably higher than those with non-cancer ulcerative colitis are SEQ ID NOs: 33, 35, 36, 43, 68-76, and 78. The CpG site in the base sequence represented by ("+" in the table). The CpG site used as a marker is not limited to these 18 CpG sites, and includes other CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, and 67-80. .
　各ＣｐＧサイトについては、予め、発癌潰瘍性大腸炎患者と非癌潰瘍性大腸炎患者を識別するための基準値を設定しておく。３２ＣｐＧセットのうち表５～７で「＋」と記されているＣｐＧサイトと３４ＣｐＧセット及び１８ＣｐＧセットのうち表８～１２で「＋」と記されているＣｐＧサイトとの場合には、測定されたメチル化率が予め設定された基準値以上である場合に、ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高い、と判定する。３２ＣｐＧセットのうち表５～７で「－」と記されているＣｐＧサイトと３４ＣｐＧセット及び１８ＣｐＧセットのうち表８～１２で「－」と記されているＣｐＧサイトとの場合には、測定されたメチル化率が予め設定された基準値以下である場合に、ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高い、と判定する。 For each CpG site, a reference value for distinguishing between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient is set in advance. Measured in the case of CpG sites marked as “+” in Tables 5-7 and CpG sites marked as “+” in Tables 8-12 out of 34 CpG sets and 18 CpG sets in 32 CpG sets When the methylation rate is equal to or higher than a preset reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer. Measured in the case of CpG sites marked as “−” in Tables 5-7 and CpG sites marked as “−” in Tables 8-12 out of 34 CpG sets and 18 CpG sets in 32 CpG sets When the methylation rate is equal to or lower than a preset reference value, it is determined that a human ulcerative colitis patient is likely to develop colorectal cancer.
　各ＣｐＧサイトの基準値は、発癌潰瘍性大腸炎患者群と非癌潰瘍性大腸炎患者群の当該ＣｐＧサイトのメチル化率を測定し、両群を区別することができる閾値として実験的に求めることができる。具体的には、任意のＣｐＧサイトのメチル化の基準値は、一般的な統計学的手法によって求められる。下記にその例を示すが、本発明における基準値の決め方はこれらに限定されるものではない。 The reference value of each CpG site is experimentally determined as a threshold value that can measure the methylation rate of the CpG site in the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group, and distinguish both groups. be able to. Specifically, the standard value for methylation of an arbitrary CpG site is obtained by a general statistical method. Examples thereof are shown below, but the method of determining the reference value in the present invention is not limited to these.
　基準値の求め方の一例としては、例えば、まず、任意のＣｐＧサイトについて、潰瘍性大腸炎患者のうち内視鏡検査での生検組織による病理検査により大腸癌と診断されなかった患者（非癌潰瘍性大腸炎患者）の直腸粘膜のＤＮＡメチル化を測定する。複数の患者について測定した後に、その平均値又は中央値などによりこれらの患者群のメチル化を代表する数値を算出し、これを基準値とすることができる。 As an example of how to obtain the reference value, for example, for any CpG site, patients who have not been diagnosed with colon cancer by pathological examination using biopsy tissue in endoscopic examination among patients with ulcerative colitis (non- DNA methylation of rectal mucosa of patients with cancer ulcerative colitis is measured. After measuring about several patients, the numerical value which represents the methylation of these patient groups by the average value or the median value, etc. can be calculated, and this can be made into a reference value.
　その他、複数の非癌潰瘍性大腸炎患者と複数の発癌潰瘍性大腸炎患者に対して、それぞれ直腸粘膜のＤＮＡメチル化を測定し、平均値又は中央値などにより発癌潰瘍性大腸炎患者群と非癌潰瘍性大腸炎患者群のメチル化を代表する数値とばらつきをそれぞれ算出した後、ばらつきも考慮して両数値が区別されるような閾値を求め、これを基準値とすることができる。 In addition, for multiple non-cancer ulcerative colitis patients and multiple carcinogenic ulcerative colitis patients, DNA methylation of the rectal mucosa was measured respectively, and the mean or median etc. After calculating a numerical value representative of methylation and variation of the non-cancer ulcerative colitis patient group, a threshold value that distinguishes both values in consideration of the variation can be obtained and used as a reference value.
　本発明においてマーカーとされるＣｐＧサイトとしては、配列番号１～１６で表される塩基配列中のＣｐＧサイトのみを用いてもよい。これらの１６個のＣｐＧサイト（以下、まとめて「１６ＣｐＧセット」ということがある。）は、３２ＣｐＧセットのうち、発癌潰瘍性大腸炎患者の大腸の非癌部位と癌部位とのメチル化率の差が小さいものである。本発明においてマーカーとされるＣｐＧサイトとしては、配列番号１～９で表される塩基配列中のＣｐＧサイトのみを用いることも好ましい。これらの９個のＣｐＧサイト（以下、まとめて「９ＣｐＧセット」ということがある。）は、１６ＣｐＧセットのうち、発癌潰瘍性大腸炎患者の大腸の非癌部位と癌部位とのメチル化率の差がより小さいものである。 As the CpG site used as a marker in the present invention, only the CpG site in the base sequence represented by SEQ ID NOs: 1 to 16 may be used. These 16 CpG sites (hereinafter, collectively referred to as “16CpG set”) are, in the 32CpG set, the methylation rate between the non-cancerous site and the cancerous site of the colon of carcinogenic ulcerative colitis patients. The difference is small. As the CpG site used as a marker in the present invention, it is also preferable to use only the CpG site in the nucleotide sequence represented by SEQ ID NOs: 1 to 9. These nine CpG sites (hereinafter, collectively referred to as “9CpG set”) are, of the 16CpG set, the methylation rate between the non-cancerous site and the cancerous site of the colon of carcinogenic ulcerative colitis patients. The difference is smaller.
　前記判定工程においては、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、３１、４５、６４、６５、６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３～１０、１３、１９、２０、２５、２８、３０、３２～４４、４６～６３、６６、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する。本発明に係る判定方法においては、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、３１、４５、６４、６５、６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３～１０、１３、１９、２０、２５、２８、３０、３２～４４、４６～６３、６６、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が２以上、好ましくは３以上、より好ましくは５以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定することにより、より精度よく判定を行うことができる。 In the determination step, it is represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, 31, 45, 64, 65, 67, 77, 79, and 80. One or more of the CpG sites in the base sequence have a methylation rate not higher than a preset reference value, or SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, 32 to When one or more of the CpG sites in the base sequences represented by 44, 46 to 63, 66, 68 to 76, and 78 have a methylation rate equal to or higher than a preset reference value, the human Determine that patients with ulcerative colitis are more likely to develop colorectal cancer. In the determination method according to the present invention, SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, 31, 45, 64, 65, 67, 77, 79, and 80 Among the CpG sites in the represented nucleotide sequence, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, 32 The sum of the number of CpG sites with a methylation rate equal to or higher than a preset reference value among CpG sites in the base sequences represented by ~ 44, 46-63, 66, 68-76 and 78 is 2 or more In the case of preferably 3 or more, more preferably 5 or more, it is possible to make a more accurate determination by determining that the human ulcerative colitis patient has a high possibility of developing colon cancer. .
　前記３２ＣｐＧセットを本発明においてマーカーとする場合、すなわち、前記測定工程において前記３２ＣｐＧセットのメチル化率を測定する場合には、前記判定工程においては、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、及び３１で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３～１０、１３、１９、２０、２５、２８、３０、及び３２で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する。本発明に係る判定方法においては、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、及び３１で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３～１０、１３、１９、２０、２５、２８、３０、及び３２で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が３以上、好ましくは５以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定することにより、より精度よく判定を行うことができる。 When the 32CpG set is used as a marker in the present invention, that is, when the methylation rate of the 32CpG set is measured in the measurement step, in the determination step, SEQ ID NOs: 1, 2, 11, 12, 14 to One or more of the CpG sites in the base sequences represented by 18, 21 to 24, 26, 27, 29, and 31 have a methylation rate equal to or lower than a preset reference value, or SEQ ID NO: When one or more CpG sites in the base sequence represented by 3 to 10, 13, 19, 20, 25, 28, 30, and 32 have a methylation rate equal to or higher than a preset reference value Furthermore, it is determined that the human ulcerative colitis patient has a high possibility of developing colorectal cancer. In the determination method according to the present invention, methylation is performed among CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, and 31. The number of CpG sites whose rate is less than or equal to a preset reference value, and methyl of the CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32 When the sum of the number of CpG sites with a conversion rate equal to or higher than a preset reference value is 3 or more, preferably 5 or more, the human ulcerative colitis patient may develop colon cancer By determining that the value is high, the determination can be performed with higher accuracy.
　前記３４ＣｐＧセットを本発明においてマーカーとする場合には、前記判定工程においては、配列番号４５、６４、及び６５で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３３～４４、４６～６３、及び６６で表される塩基配列中のＣｐＧサイトのうち少１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する。本発明に係る判定方法においては、配列番号４５、６４、及び６５で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３３～４４、４６～６３、及び６６で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が２以上、好ましくは３以上、より好ましくは５以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定することにより、より精度よく判定を行うことができる。 When the 34CpG set is used as a marker in the present invention, in the determination step, one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65 have a methylation rate. The methylation rate is preset for at least one of the CpG sites in the base sequence represented by SEQ ID NOs: 33-44, 46-63, and 66, which are below the preset reference value. If it is equal to or higher than the reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer. In the determination method according to the present invention, among the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and the sequence Among the CpG sites in the base sequences represented by the numbers 33 to 44, 46 to 63, and 66, the sum with the number of CpG sites whose methylation rate is not less than a preset reference value is preferably 2 or more, preferably 3 As described above, when it is more preferably 5 or more, it can be determined with higher accuracy by determining that the human ulcerative colitis patient has a high possibility of developing colorectal cancer.
　前記１８ＣｐＧセットを本発明においてマーカーとする場合には、前記判定工程においては、配列番号６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３３、３５、３６、４３、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する。本発明に係る判定方法においては、配列番号６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３３、３５、３６、４３、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が２以上、好ましくは３以上、より好ましくは５以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定することにより、より精度よく判定を行うことができる。 When the 18CpG set is used as a marker in the present invention, in the determination step, one or more CpG sites in the base sequence represented by SEQ ID NOs: 67, 77, 79, and 80 are methylated. The rate is below a preset reference value, or one or more of the CpG sites in the base sequence represented by SEQ ID NOs: 33, 35, 36, 43, 68 to 76, and 78 are methylated When the rate is equal to or higher than a preset reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer. In the determination method according to the present invention, among the CpG sites in the base sequences represented by SEQ ID NOs: 67, 77, 79, and 80, the number of CpG sites having a methylation rate equal to or lower than a preset reference value And the sum of the number of CpG sites having a methylation rate equal to or higher than a preset reference value among the CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, 68 to 76, and 78. When it is 2 or more, preferably 3 or more, more preferably 5 or more, it is determined that the human ulcerative colitis patient has a high possibility of developing colorectal cancer, thereby making a more accurate determination Can do.
　本発明においては、配列番号１～８０で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトをマーカーとして用いることができる。本発明においてマーカーとするＣｐＧサイトとしては、配列番号１～８０で表される塩基配列中の括弧内の８０個のＣｐＧサイトの全て（以下、まとめて「８０ＣｐＧセット」ということがある。）であってもよく、前記３２ＣｐＧセットであってもよく、前記１６ＣｐＧセットであってもよく、前記９ＣｐＧセットであってもよく、前記３４ＣｐＧセットであってもよく、前記１８ＣｐＧセットであってもよい。前記３２ＣｐＧセットのＣｐＧサイト、前記１６ＣｐＧセットのＣｐＧサイト、及び前記９ＣｐＧセットのＣｐＧサイトは、いずれも、非癌潰瘍性大腸炎患者群と発癌潰瘍性大腸炎患者群のメチル化率の分散が小さく、非癌潰瘍性大腸炎患者群と発癌潰瘍性大腸炎患者群の識別能が高い点で優れている。一方で、前記３４ＣｐＧセット及び前記１８ＣｐＧセットは、前記３２ＣｐＧセット、前記１６ＣｐＧセットのＣｐＧサイト、及び前記９ＣｐＧセットのＣｐＧサイトに比べて特異度はやや低くなるものの、感度が非常に高く、例えば、発癌潰瘍性大腸炎の一次スクリーニング検査には非常に好適である。 In the present invention, one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 can be used as markers. The CpG sites used as markers in the present invention are all 80 CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 1 to 80 (hereinafter sometimes collectively referred to as “80 CpG set”). May be the 32CpG set, the 16CpG set, the 9CpG set, the 34CpG set, or the 18CpG set. In the CpG site of the 32CpG set, the CpG site of the 16CpG set, and the CpG site of the 9CpG set, all of the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group have small dispersion of the methylation rate. The non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group are excellent in that they have a high discrimination ability. On the other hand, although the 34CpG set and the 18CpG set are slightly less specific than the CpG site of the 32CpG set, the 16CpG set, and the CpG site of the 9CpG set, the sensitivity is very high. It is very suitable for primary screening test for ulcerative colitis.
　本発明に係る判定方法のうち、特定のＤＭＲの平均メチル化率の値自体に基づいて判定を行う方法は、具体的には、ヒト潰瘍性大腸炎患者の大腸癌発症可能性を判定する方法であって、ヒト潰瘍性大腸炎患者から採取された生体試料から回収されたＤＮＡ中の、本発明においてマーカーとする特定のＤＭＲ中に存在する１個以上のＣｐＧサイトのメチル化率を測定する測定工程と、前記測定工程において測定されたメチル化率に基づいて算出されたＤＭＲの平均メチル化率と、各ＤＭＲの平均メチル化率に対して予めそれぞれ設定された基準値に基づいて、前記ヒト潰瘍性大腸炎患者の大腸癌発症の可能性を判定する判定工程を有する。なお、各ＤＭＲの平均メチル化率は、当該ＤＭＲ中のＣｐＧサイトのうち、前記測定工程においてメチル化率が測定された全てのＣｐＧサイトのメチル化率の平均値として算出する。 Among the determination methods according to the present invention, the method of determining based on the value of the average methylation rate of a specific DMR is specifically a method of determining the likelihood of developing colorectal cancer in a human ulcerative colitis patient The methylation rate of one or more CpG sites present in a specific DMR used as a marker in the present invention in DNA collected from a biological sample collected from a patient with human ulcerative colitis is measured. Based on the measurement step, the average methylation rate of the DMR calculated based on the methylation rate measured in the measurement step, and a reference value set in advance for the average methylation rate of each DMR, A determination step of determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis. The average methylation rate of each DMR is calculated as the average value of the methylation rates of all CpG sites whose methylation rates were measured in the measurement step among the CpG sites in the DMR.
　本発明においてマーカーとされるＤＭＲは、具体的には、ＤＭＲ番号１～１１２で表されるＤＭＲからなる群より選択される１か所以上のＤＭＲである。各ＤＭＲの染色体上の位置及び対応する遺伝子を表１３～１６に示す。表中のＤＭＲの開始点と終点の塩基位置は、ヒトゲノム配列のデータセット「ＧＲＣｈ３７／ｈｇ１９」に基づく。これらのＤＭＲ中に存在するＣｐＧサイトを含む塩基配列を有するＤＮＡ断片は、潰瘍性大腸炎患者の大腸癌発症可能性を判定するためのＤＮＡメチル化率分析用マーカーとして用いることができる。 The DMR used as a marker in the present invention is specifically one or more DMRs selected from the group consisting of DMRs represented by DMR numbers 1-112. The position of each DMR on the chromosome and the corresponding gene are shown in Tables 13-16. The base positions of the start point and end point of DMR in the table are based on the human genome sequence data set “GRCh37 / hg19”. These DNA fragments having a base sequence containing a CpG site present in DMR can be used as a DNA methylation rate analysis marker for determining the possibility of developing colon cancer in patients with ulcerative colitis.
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
　ＤＭＲ番号１～１１２で表されるＤＭＲ（以下、まとめて「１１２ＤＭＲセット」ということがある。）は、各領域中に含まれる複数のＣｐＧサイトのメチル化率が、非癌潰瘍性大腸炎患者群と発癌潰瘍性大腸炎患者群とで大きく相違するものである。このうち、発癌潰瘍性大腸炎患者のＤＭＲの平均メチル化率（ＤＭＲ中に存在している複数のＣｐＧサイトのメチル化率の平均値）が非癌潰瘍性大腸炎患者よりもかなり低いものが、ＤＭＲ番号１、３～２０、２３～２８、３１～４６、４９～６０、６２、６５～６９、７１、７３、７４、７９、８１、８２、８４、８６、８７、９０～９２、９５、１０１、１０３、１０９、１１０、及び１１２で表されるＤＭＲであり（表中、「－」）、発癌潰瘍性大腸炎患者のＤＭＲの平均メチル化率が非癌潰瘍性大腸炎患者よりもかなり高いものが、ＤＭＲ番号２、２１、２２、２９、３０、４７、４８、６１，６３、６４、７０、７２、７５～７８、８０、８３、８５、８８、８９、９３、９４、９６～１００、１０２、１０４～１０８、及び１１１で表されるＤＭＲである（表中、「＋」）。 DMRs represented by DMR numbers 1-112 (hereinafter sometimes referred to collectively as “112 DMR sets”) have a plurality of CpG site methylation rates in patients with non-cancer ulcerative colitis. Group and carcinogenic ulcerative colitis patient group are greatly different. Among them, the average methylation rate of DMR (average value of methylation rates of a plurality of CpG sites present in DMR) of carcinogenic ulcerative colitis patients is considerably lower than that of non-cancer ulcerative colitis patients. , DMR numbers 1, 3 to 20, 23 to 28, 31 to 46, 49 to 60, 62, 65 to 69, 71, 73, 74, 79, 81, 82, 84, 86, 87, 90 to 92, 95 , 101, 103, 109, 110, and 112 ("-" in the table), and the average methylation rate of DMR in patients with carcinogenic ulcerative colitis is higher than that in patients with non-cancer ulcerative colitis The fairly high ones are DMR numbers 2, 21, 22, 29, 30, 47, 48, 61, 63, 64, 70, 72, 75-78, 80, 83, 85, 88, 89, 93, 94, 96 ~ 100, 102, 104-108, and 11 In a DMR represented (in the table, "+").
　本発明において、ＤＭＲの平均メチル化率をマーカーとする場合には、ＤＭＲ番号１～１１２で表されるＤＭＲのうちの１か所をマーカーとしてもよく、ＤＭＲ番号１～１１２で表されるＤＭＲからなる群より選択される任意の２か所以上をマーカーとしてもよく、ＤＭＲ番号１～１１２で表されるＤＭＲの全てをマーカーとしてもよい。本発明においては、判定精度をより高められることから、ＤＭＲ番号１～１１２で表されるＤＭＲからなる群のうちマーカーとして用いるＤＭＲは、２か所以上であることが好ましく、３か所以上であることがより好ましく、４か所以上であることがさらに好ましく、５か所以上であることがよりさらに好ましい。 In the present invention, when the average methylation rate of DMR is used as a marker, one of the DMRs represented by DMR numbers 1-112 may be used as a marker, and the DMR represented by DMR numbers 1-112 Any two or more locations selected from the group consisting of may be used as markers, and all of the DMRs represented by DMR numbers 1-112 may be used as markers. In the present invention, since the determination accuracy can be further improved, the number of DMRs used as markers in the group consisting of DMRs represented by DMR numbers 1-112 is preferably 2 or more, and more than 3 More preferably, it is more preferably 4 or more, and still more preferably 5 or more.
　より高い判定精度が得られることから、本発明においてそのメチル化率がマーカーとして用いられるＤＭＲは、ＤＭＲ番号１～５８で表されるＤＭＲ（以下、まとめて「５８ＤＭＲセット」ということがある。）からなる群より選択される１か所以上であることが好ましく、５８ＤＭＲセットから選択される２か所以上であることがより好ましく、５８ＤＭＲセットから選択される３か所以上であることがさらに好ましく、５８ＤＭＲセットから選択される４か所以上であることがよりさらに好ましく、５８ＤＭＲセットから選択される５か所以上であることが特に好ましい。なかでも、ＤＭＲ番号１～１１で表されるＤＭＲ（以下、まとめて「１１ＤＭＲセット」ということがある。）からなる群より選択される１か所以上であることが好ましく、１１ＤＭＲセットから選択される２か所以上であることがより好ましく、１１ＤＭＲセットから選択される３か所以上であることがさらに好ましく、１１ＤＭＲセットから選択される４か所以上であることがよりさらに好ましく、１１ＤＭＲセットから選択される５か所以上であることが特に好ましい。 Since higher determination accuracy can be obtained, the DMR whose methylation rate is used as a marker in the present invention is a DMR represented by DMR numbers 1 to 58 (hereinafter sometimes referred to as “58 DMR set”). 1 or more selected from the group consisting of, more preferably 2 or more selected from the 58 DMR set, more preferably 3 or more selected from the 58 DMR set. 4 or more selected from the 58 DMR set, and more preferably 5 or more selected from the 58 DMR set. Among these, it is preferable that the number is one or more selected from the group consisting of DMRs represented by DMR numbers 1 to 11 (hereinafter sometimes collectively referred to as “11 DMR sets”), and selected from 11 DMR sets. 2 or more, more preferably 3 or more selected from the 11 DMR set, even more preferably 4 or more selected from the 11 DMR set, from the 11 DMR set. It is particularly preferable that the number is 5 or more selected.
　各ＤＭＲの平均メチル化率は、それぞれのＤＭＲ中に含まれる全てのＣｐＧサイトのメチル化率の平均値としてもよく、それぞれのＤＭＲ中に含まれる全てのＣｐＧサイトから少なくとも１個のＣｐＧサイトを任意に選出し、この選出されたＣｐＧサイトのメチル化率の平均値としてもよい。各ＣｐＧサイトのメチル化率は、配列番号１等で表される塩基配列中のＣｐＧサイトのメチル化率の測定と同様にして測定することができる。 The average methylation rate of each DMR may be an average value of the methylation rates of all CpG sites included in each DMR, and at least one CpG site is selected from all CpG sites included in each DMR. It is good also as selecting arbitrarily and making it the average value of the methylation rate of this selected CpG site. The methylation rate of each CpG site can be measured in the same manner as the measurement of the methylation rate of the CpG site in the base sequence represented by SEQ ID NO: 1 or the like.
　各ＤＭＲの平均メチル化率については、予め、発癌潰瘍性大腸炎患者と非癌潰瘍性大腸炎患者を識別するための基準値を設定しておく。前記１１２ＤＭＲセットのうち表１３～１６で「＋」と記されているＤＭＲの場合には、測定されたＤＭＲの平均メチル化率が予め設定された基準値以上である場合に、ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高い、と判定する。１１２ＤＭＲセットのうち表１３～１６で「－」と記されているＤＭＲの場合には、測定されたＤＭＲの平均メチル化率が予め設定された基準値以下である場合に、ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高い、と判定する。 For the average methylation rate of each DMR, a reference value for distinguishing between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient is set in advance. In the case of the DMR marked “+” in Tables 13 to 16 in the 112 DMR set, when the average methylation rate of the measured DMR is equal to or higher than a preset reference value, It is determined that the patient with inflammation is likely to develop colorectal cancer. In the case of a DMR marked as “-” in Tables 13 to 16 in the 112 DMR set, human ulcerative colitis when the average methylation rate of the measured DMR is not more than a preset reference value It is determined that the patient is likely to have colorectal cancer.
　各ＤＭＲの平均メチル化率の基準値は、発癌潰瘍性大腸炎患者群と非癌潰瘍性大腸炎患者群の当該ＤＭＲの平均メチル化率を測定し、両群を区別することができる閾値として実験的に求めることができる。具体的には、ＤＭＲの平均メチル化率の基準値は、一般的な統計学的手法によって求められる。 The standard value of the average methylation rate of each DMR is a threshold value that can be used to measure the average methylation rate of the DMR of the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group, and to distinguish the two groups. It can be determined experimentally. Specifically, the standard value of the average methylation rate of DMR is obtained by a general statistical method.
　前記８０ＣｐＧセット等のＣｐＧサイトのメチル化率をマーカーとする場合には、本発明に係る判定方法は、前記判定工程を、前記測定工程において測定されたメチル化率と、予め設定された多変量判別式に基づいて、前記ヒト潰瘍性大腸炎患者の大腸癌発症の可能性を判定することができる。当該多変量判別式は、前記配列番号１～８０で表される塩基配列中のＣｐＧサイトのうち１か所以上のＣｐＧサイトのメチル化率を変数として含む。 When the methylation rate of the CpG site such as the 80CpG set is used as a marker, the determination method according to the present invention includes the determination step including a methylation rate measured in the measurement step and a preset multivariate. Based on the discriminant, the possibility of developing colorectal cancer in the human ulcerative colitis patient can be determined. The multivariate discriminant includes, as a variable, the methylation rate of one or more CpG sites among the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80.
　前記１１２ＤＭＲセットからなる群より選択される１か所以上のＤＭＲの平均メチル化率をマーカーとする場合には、本発明に係る判定方法は、前記判定工程を、前記測定工程において測定されたメチル化率に基づいて算出されたＤＭＲの平均メチル化率と、予め設定された多変量判別式に基づいて、前記ヒト潰瘍性大腸炎患者の大腸癌発症の可能性を判定することができる。当該多変量判別式は、前記１１２ＤＭＲセット中のＣｐＧサイトのうち１か所以上のＣｐＧサイトのメチル化率を変数として含む。 In the case where the average methylation rate of one or more DMRs selected from the group consisting of the 112 DMR sets is used as a marker, the determination method according to the present invention includes the determination step including the methylation measured in the measurement step. Based on the average methylation rate of DMR calculated based on the conversion rate and a multivariate discriminant set in advance, the possibility of the onset of colon cancer in the human ulcerative colitis patient can be determined. The multivariate discriminant includes, as a variable, the methylation rate of one or more CpG sites among the CpG sites in the 112DMR set.
　本発明において用いられる多変量判別式は、２群を判別するために用いられる一般的な手法により求めることができる。当該多変量判別式としては、例えば、ロジスティック回帰式、線形判別式、ナイーブベイズ分類器で作成された式、又はサポートベクターマシンで作成された式が挙げられるが、これらに限定されるものではない。これらの多変量判別式は、例えば、発癌潰瘍性大腸炎患者群と非癌潰瘍性大腸炎患者群について、前記配列番号１～８０で表される塩基配列中のＣｐＧサイトのうち１か所又は２か所以上のＣｐＧサイトのメチル化率を測定し、得られたメチル化率を変数として、常法により作成することができる。また、発癌潰瘍性大腸炎患者群と非癌潰瘍性大腸炎患者群について、前記１１２ＤＭＲセット中のＤＭＲのうち１か所又は２か所以上のＤＭＲの平均メチル化率を測定し、得られたメチル化率を変数として、常法により作成することができる。 The multivariate discriminant used in the present invention can be obtained by a general method used to discriminate between the two groups. Examples of the multivariate discriminant include, but are not limited to, a logistic regression equation, a linear discriminant, a formula created with a naive Bayes classifier, or a formula created with a support vector machine. . These multivariate discriminants include, for example, one of the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 for the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group or The methylation rate of two or more CpG sites can be measured, and the resulting methylation rate can be used as a variable to prepare a standard method. In addition, for the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group, the average methylation rate of one or more DMRs among the DMRs in the 112 DMR set was measured and obtained. The methylation rate can be used as a variable, and can be prepared by a conventional method.
　本発明において用いられる多変量判別式には、予め、発癌潰瘍性大腸炎患者と非癌潰瘍性大腸炎患者を識別するための基準判別値を設定しておく。基準判別値は、発癌潰瘍性大腸炎患者群と非癌潰瘍性大腸炎患者群について、使用する多変量判別式の値である判別値を求め、発癌潰瘍性大腸炎患者群の判別値と非癌潰瘍性大腸炎患者群の判別値とを比較し、両群を区別することができる閾値として実験的に求めることができる。 In the multivariate discriminant used in the present invention, a reference discriminating value for discriminating between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient is set in advance. For the standard discriminant value, the discriminant value, which is the value of the multivariate discriminant used for the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group, is obtained. It can be experimentally determined as a threshold value for distinguishing both groups by comparing the discriminant values of the cancer ulcerative colitis patient group.
　多変量判別式を用いて判定を行う場合、具体的には、前記測定工程において、使用する多変量判別式がそのメチル化率を変数として含むＣｐＧサイトのメチル化率又はＤＭＲの平均メチル化率を測定し、前記判定工程において、前記測定工程において測定されたメチル化率と前記多変量判別式に基づいて当該多変量判別式の値である判別値を算出し、この判別値と予め設定した基準判別値とに基づいて、ＣｐＧサイトのメチル化率又はＤＭＲの平均メチル化率が測定されたヒト潰瘍性大腸炎患者が大腸癌を発症している可能性の高低を判定する。当該判別値が予め設定された基準判別値以上である場合に、当該ヒト潰瘍性大腸炎患者が、大腸癌を発症している可能性が高いと判定する。 When the determination is made using the multivariate discriminant, specifically, in the measurement step, the methylation rate of the CpG site or the average methylation rate of the DMR in which the multivariate discriminant used includes the methylation rate as a variable. In the determination step, a discriminant value which is a value of the multivariate discriminant is calculated based on the methylation rate measured in the measurement step and the multivariate discriminant, and the discriminant value is set in advance. Based on the reference discriminant value, it is determined whether a human ulcerative colitis patient whose CpG site methylation rate or DMR average methylation rate has been measured has developed colorectal cancer. When the discriminant value is greater than or equal to a preset reference discriminant value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
　本発明において用いられる多変量判別式としては、前記３４ＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を変数として含む式が好ましく、前記３４ＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率のみを変数として含む式がより好ましく、前記３４ＣｐＧサイトからなる群より任意に選択される２～１０か所のＣｐＧサイトのメチル化率のみを変数として含む式がさらに好ましく、前記３４ＣｐＧサイトからなる群より任意に選択される２～５か所のＣｐＧサイトのメチル化率のみを変数として含む式がよりさらに好ましい。 The multivariate discriminant used in the present invention is preferably a formula containing as a variable the methylation rate of one or more CpG sites selected from the group consisting of the 34 CpG sites, and selected from the group consisting of the 34 CpG sites. More preferably, the formula includes only the methylation rate of one or more CpG sites as a variable, and only the methylation rate of 2 to 10 CpG sites arbitrarily selected from the group consisting of the 34 CpG sites is used as a variable. More preferably, the formula includes only the methylation rate of 2 to 5 CpG sites arbitrarily selected from the group consisting of the 34 CpG sites as a variable.
　本発明において用いられる多変量判別式としては、前記１８ＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を変数として含む式が好ましく、前記１８ＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率のみを変数として含む式がより好ましく、前記１８ＣｐＧサイトからなる群より任意に選択される２～１０か所のＣｐＧサイトのメチル化率のみを変数として含む式がさらに好ましく、前記１８ＣｐＧサイトからなる群より任意に選択される２～５か所のＣｐＧサイトのメチル化率のみを変数として含む式がよりさらに好ましい。 The multivariate discriminant used in the present invention is preferably a formula containing as a variable the methylation rate of one or more CpG sites selected from the group consisting of the 18CpG sites, and selected from the group consisting of the 18CpG sites. More preferably, the formula includes only the methylation rate of one or more CpG sites as a variable, and only the methylation rate of 2 to 10 CpG sites arbitrarily selected from the group consisting of the 18 CpG sites is used as a variable. More preferably, the formula includes only the methylation rate of 2 to 5 CpG sites arbitrarily selected from the group consisting of the 18 CpG sites as a variable.
　前記３４ＣｐＧセット及び前記１８ＣｐＧセットを構成するＣｐＧサイトは、これらのセットから任意で２～１０個、好ましくは２～５個のＣｐＧサイトを選択し、この選択されたＣｐＧサイトのみを用いた場合でも、充分な感度及び特異度で、ヒト潰瘍性大腸炎患者の大腸癌発症可能性を判定することができる。例えば、後記実施例２に示す通り、前記３４ＣｐＧセットのうち、配列番号３４で表される塩基配列中のＣｐＧサイト、配列番号３７で表される塩基配列中のＣｐＧサイト、及び配列番号５６で表される塩基配列中のＣｐＧサイトの３個のＣｐＧサイトをマーカーとして用い、これらの３個のＣｐＧサイトのメチル化率を変数としてロジスティック回帰により作成された多変量判別式を用いた場合には、ヒト潰瘍性大腸炎患者について、感度約９６％、特異度約９２％で大腸癌の発症可能性を判定することができる。臨床検査等において、メチル化率を測定するＣｐＧサイトの数が多いと、労力とコストが過大となるおそれがある。マーカーとするＣｐＧサイトを前記３４ＣｐＧセット及び前記１８ＣｐＧセットを構成するＣｐＧサイトから選抜することにより、２～１０個という、臨床検査において測定可能な妥当な数のＣｐＧサイトで、精度よくヒト潰瘍性大腸炎患者の大腸癌発症可能性を判定することができる。 The CpG sites constituting the 34 CpG set and the 18 CpG set are arbitrarily selected from 2 to 10, preferably 2 to 5 CpG sites from these sets, and even when only the selected CpG sites are used. With sufficient sensitivity and specificity, it is possible to determine the possibility of developing colon cancer in human ulcerative colitis patients. For example, as shown in Example 2 below, among the 34 CpG sets, the CpG site in the base sequence represented by SEQ ID NO: 34, the CpG site in the base sequence represented by SEQ ID NO: 37, and the sequence represented by SEQ ID NO: 56 In the case of using a multivariate discriminant created by logistic regression using the three CpG sites of the CpG site in the base sequence to be used as markers and the methylation rate of these three CpG sites as variables, For human ulcerative colitis patients, the likelihood of developing colon cancer can be determined with a sensitivity of about 96% and a specificity of about 92%. In clinical examinations and the like, if the number of CpG sites for measuring the methylation rate is large, labor and cost may be excessive. By selecting a CpG site as a marker from the CpG sites constituting the 34CpG set and the 18CpG set, it is an appropriate number of CpG sites that can be measured in clinical examinations, and is accurate to human ulcerative colon. The possibility of developing colorectal cancer in patients with inflammation can be determined.
　本発明において用いられる多変量判別式としては、前記１１２ＤＭＲセットからなる群より選択される１か所以上のＤＭＲの平均メチル化率を変数として含む式が好ましく、前記１１２ＤＭＲセットからなる群より選択される２か所以上のＤＭＲの平均メチル化率のみを変数として含む式がより好ましく、前記１１２ＤＭＲセットからなる群より任意に選択される３か所以上のＤＭＲの平均メチル化率のみを変数として含む式がさらに好ましく、前記１１２ＤＭＲセットからなる群より任意に選択される４か所以上のＤＭＲの平均メチル化率のみを変数として含む式がよりさらに好ましく、前記１１２ＤＭＲセットからなる群より任意に選択される５か所以上のＤＭＲの平均メチル化率のみを変数として含む式が特に好ましい。中でも、前記５８ＤＭＲセットからなる群より選択される１か所以上のＤＭＲの平均メチル化率を変数として含む式が好ましく、前記５８ＤＭＲセットからなる群より選択される２か所以上のＤＭＲの平均メチル化率のみを変数として含む式がより好ましく、前記５８ＤＭＲセットからなる群より任意に選択される２～１０か所のＤＭＲの平均メチル化率のみを変数として含む式がさらに好ましく、前記５８ＤＭＲセットからなる群より任意に選択される３～１０か所のＤＭＲの平均メチル化率のみを変数として含む式がよりさらに好ましく、前記５８ＤＭＲセットからなる群より任意に選択される５～１０か所のＤＭＲの平均メチル化率のみを変数として含む式が特に好ましい。より好ましくは、前記１１ＤＭＲセットからなる群より選択される１か所以上のＤＭＲの平均メチル化率を変数として含む式が好ましく、前記１１ＤＭＲセットからなる群より選択される２か所以上のＤＭＲの平均メチル化率のみを変数として含む式がより好ましく、前記１１ＤＭＲセットからなる群より任意に選択される２～１０か所のＤＭＲの平均メチル化率のみを変数として含む式がさらに好ましく、前記１１ＤＭＲセットからなる群より任意に選択される３～１０か所のＤＭＲの平均メチル化率のみを変数として含む式がよりさらに好ましく、前記１１ＤＭＲセットからなる群より任意に選択される５～１０か所のＤＭＲの平均メチル化率のみを変数として含む式が特に好ましい。 The multivariate discriminant used in the present invention is preferably an expression including, as a variable, an average methylation rate of one or more DMRs selected from the group consisting of the 112 DMR set, and selected from the group consisting of the 112 DMR set. More preferably, the formula includes only the average methylation rate of two or more DMRs as a variable, and includes only the average methylation rate of three or more DMRs arbitrarily selected from the group consisting of the 112 DMR sets as a variable. More preferably, the formula includes only the average methylation rate of four or more DMRs arbitrarily selected from the group consisting of the 112 DMR set as a variable, and more preferably the formula is arbitrarily selected from the group consisting of the 112 DMR set. A formula including only the average methylation rate of 5 or more DMRs as a variable is particularly preferable. Among them, a formula including an average methylation rate of one or more DMRs selected from the group consisting of the 58 DMR sets as a variable is preferable, and an average methyl of two or more DMRs selected from the group consisting of the 58 DMR sets is preferable. More preferably, the formula includes only the conversion rate as a variable, and more preferably includes only the average methylation rate of 2 to 10 DMRs arbitrarily selected from the group consisting of the 58 DMR set as a variable. More preferably, the formula includes only the average methylation rate of 3 to 10 DMRs arbitrarily selected from the group consisting of the variables, and 5 to 10 DMRs arbitrarily selected from the group consisting of the 58 DMR sets. Particularly preferred is a formula containing only the average methylation rate of as a variable. More preferably, a formula including, as a variable, an average methylation rate of one or more DMRs selected from the group consisting of the 11 DMR sets is preferable, and two or more DMRs selected from the group consisting of the 11 DMR sets are used. A formula including only the average methylation rate as a variable is more preferable, and a formula including only the average methylation rate of 2 to 10 DMRs arbitrarily selected from the group consisting of the 11 DMR sets as a variable is more preferable. More preferably, the formula includes only the average methylation rate of 3 to 10 DMRs arbitrarily selected from the group consisting of the set as a variable, and 5 to 10 sites arbitrarily selected from the group consisting of the 11 DMR sets. Particularly preferred is a formula containing only the average methylation rate of DMR as a variable.
　本発明に係る判定方法に供される生体試料は、ヒト潰瘍性大腸炎患者から採取された生体試料であって、当該患者のゲノムＤＮＡが含まれているものであれば特に限定されるものではなく、血液、血漿、血清、涙液、唾液等であってもよく、消化管粘膜や、肝臓等のその他の組織から採取された組織片であってもよい。本発明に係る判定方法に供される生体試料としては、大腸の状態をより強く反映していることから、大腸粘膜であることが好ましく、比較的低侵襲に採取可能であることから直腸粘膜であることがより好ましい。大腸の直腸粘膜は、例えば、後記の大腸粘膜採取用キットを用いて簡便に採取することができる。 The biological sample used in the determination method according to the present invention is a biological sample collected from a patient with human ulcerative colitis and is not particularly limited as long as it contains the genomic DNA of the patient. Alternatively, blood, plasma, serum, tears, saliva, or the like may be used, or tissue pieces collected from other tissues such as the digestive tract mucosa or the liver may be used. The biological sample used in the determination method according to the present invention is preferably the large intestine mucosa because it more strongly reflects the state of the large intestine, and can be collected in the rectal mucosa because it can be collected with relatively low invasiveness. More preferably. The rectal mucosa of the large intestine can be easily collected using, for example, a kit for collecting large intestine mucosa described later.
　また、当該生体試料は、ＤＮＡが抽出可能な状態であればよく、各種前処理が施されているものであってもよい。例えば、ホルマリン固定パラフィン包埋（ＦＦＰＥ）組織であってもよい。生体試料からのＤＮＡの抽出は常法により行うことができ、各種市販のＤＮＡ抽出・精製キットを使用することもできる。 In addition, the biological sample may be in a state where DNA can be extracted, and may be subjected to various pretreatments. For example, it may be a formalin fixed paraffin embedded (FFPE) tissue. Extraction of DNA from a biological sample can be performed by a conventional method, and various commercially available DNA extraction / purification kits can also be used.
　ＣｐＧサイトのメチル化率を測定する方法としては、特定のＣｐＧサイトについてメチル化シトシン塩基とメチル化されていないシトシン塩基を区別して定量可能な方法であれば、特に限定されるものではない。当該技術分野において公知の方法をそのまま又は必要に応じて適宜改変することによりＣｐＧサイトのメチル化率を測定できる。ＣｐＧサイトのメチル化率の測定方法としては、例えば、バイサルファイトシーケンス法、ＣＯＢＲＡ（Ｃｏｍｂｉｎｅｄ　Ｂｉｓｕｌｆｉｔｅ　Ｒｅｓｔｒｉｃｔｉｏｎ　Ａｎａｌｙｓｉｓ）法、ｑＡＭＰ（ｑｕａｎｔｉｔａｔｉｖｅ　ａｎａｌｙｓｉｓ　ｏｆ　ＤＮＡ　ｍｅｔｈｙｌａｔｉｏｎ　ｕｓｉｎｇ　ｒｅａｌ－ｔｉｍｅ　ＰＣＲ）法等が挙げられる。その他、ＭＩＡＭ（Ｍｉｃｒｏａｒｒａｙ－ｂａｓｅｄ　Ｉｎｔｅｇｒａｔｅｄ　Ａｎａｌｙｓｉｓ　ｏｆ　Ｍｅｔｈｙｌａｔｉｏｎ　ｂｙ　Ｉｓｏｓｃｈｉｚｏｍｅｒｓ）法を用いて行ってもよい。 The method for measuring the methylation rate of a CpG site is not particularly limited as long as it is a method capable of distinguishing and quantifying a methylated cytosine base and an unmethylated cytosine base for a specific CpG site. The methylation rate of the CpG site can be measured by appropriately modifying methods known in the art as they are or as necessary. Examples of methods for measuring the methylation rate of CpG sites include bisulfite sequencing, COBRA (Combined Bisulfite Restoration Analysis), and qAMP (quantitative analysis of DNA methylation using real-time PCR). In addition, you may perform using the MIAM (Microarray-based Integrated Analysis of Methylation by Isoschizomers) method.
＜大腸粘膜採取用キット＞
　本発明に係る大腸粘膜採取用キットは、直腸粘膜を挟んで採取するための採取具と、肛門を拡張し、当該採取具を肛門から大腸粘膜表面へ到達させるための採取補助具とを備える。以下、図１～５を参照しながら、本発明に係る大腸粘膜採取用キットについて説明する。 <Colonial mucosa collection kit>
The kit for collecting large intestine mucosa according to the present invention comprises a collection tool for collecting with the rectal mucosa sandwiched therein, and a collection aid for expanding the anus and allowing the collection tool to reach the surface of the large intestine mucosa from the anus. The colonic mucosa collection kit according to the present invention will be described below with reference to FIGS.
　図１（Ａ）～図１（Ｃ）は大腸粘膜採取用キット１の採取具２の一実施態様である採取具２Ａの説明図である。図１（Ａ）は採取具２Ａの第１の挟持片３ａと第２の挟持片３ｂが加力されていない状態の斜視図であり、図１（Ｂ）は加力された状態の斜視図である。また、図１（Ｃ）は採取具２Ａの挟持面を有する先端部の一部拡大図である。図１（Ａ）に示すように、採取具２Ａは、第１の挟持片３ａと、第２の挟持片３ｂと、連結部４と、第１の挟持面５ａと、第２の挟持面５ｂと、を有する。 FIG. 1 (A) to FIG. 1 (C) are explanatory views of a sampling tool 2A, which is an embodiment of the sampling tool 2 of the colonic mucosa sampling kit 1. FIG. FIG. 1 (A) is a perspective view of a state in which the first holding piece 3a and the second holding piece 3b of the sampling tool 2A are not applied, and FIG. 1 (B) is a perspective view of the applied state. It is. FIG. 1C is a partially enlarged view of the distal end portion having the clamping surface of the sampling tool 2A. As shown in FIG. 1 (A), the sampling tool 2A includes a first clamping piece 3a, a second clamping piece 3b, a connecting portion 4, a first clamping surface 5a, and a second clamping surface 5b. And having.
　第１の挟持片３ａは、一方の端部に大腸粘膜を挟持する第１の挟持面５ａが形成されている板状の部材であり、第２の挟持片３ｂは、一方の端部に大腸粘膜を挟持する第２の挟持面５ｂが形成されている板状の部材である。第１の挟持片３ａと第２の挟持片３ｂは、連結部４において、互いに対向した状態で、第１の挟持面５ａ及び第２の挟持面５ｂが形成されていない端部において連結されている。第１の挟持片３ａ及び第２の挟持片３ｂの形状は、板状の他、棒状であってもよく、直腸粘膜を挟んで採取するための一定の長さを有するように形成されていれば形状にこだわらない。 The first clamping piece 3a is a plate-like member in which a first clamping surface 5a for clamping the colonic mucosa is formed at one end, and the second clamping piece 3b is a large intestine at one end. It is a plate-like member on which a second clamping surface 5b that clamps the mucous membrane is formed. The first sandwiching piece 3a and the second sandwiching piece 3b are connected to each other at the end where the first sandwiching surface 5a and the second sandwiching surface 5b are not formed in the connecting portion 4 in a state of facing each other. Yes. The shape of the first sandwiching piece 3a and the second sandwiching piece 3b may be plate-like or rod-like, and may be formed to have a certain length for collecting the rectal mucosa. Don't stick to the shape.
　第１の挟持片３ａと第２の挟持片３ｂへの加力により、両者は互いに近接する。このため、採取具２Ａの第１の挟持面５ａと第２の挟持面５ｂを大腸粘膜に接触させた状態で、第１の挟持片３ａと第２の挟持片３ｂに加力することによって、第１の挟持面５ａと第２の挟持面５ｂにより大腸粘膜を挟持することができる。より詳細には、第１の挟持面５ａの辺縁部６ａと第２の挟持面５ｂの辺縁部６ｂが大腸粘膜を挟んだ状態で接する。この状態で採取具２Ａを大腸粘膜から離すことにより、第１の挟持面５ａと第２の挟持面５ｂに挟持された大腸粘膜が引きちぎられて採取される。 Both are close to each other by the force applied to the first sandwiching piece 3a and the second sandwiching piece 3b. For this reason, by applying force to the first clamping piece 3a and the second clamping piece 3b in a state where the first clamping surface 5a and the second clamping surface 5b of the sampling tool 2A are in contact with the large intestine mucosa, The large intestine mucosa can be clamped by the first clamping surface 5a and the second clamping surface 5b. More specifically, the edge 6a of the first clamping surface 5a and the edge 6b of the second clamping surface 5b are in contact with the large intestine mucosa sandwiched therebetween. In this state, by separating the collection tool 2A from the large intestine mucosa, the large intestine mucosa sandwiched between the first clamping surface 5a and the second clamping surface 5b is torn and collected.
　第１の挟持片３ａと第２の挟持片３ｂの長さは、５０～２５０ｍｍが好ましく、１００～２００ｍｍがより好ましく、７０～２００ｍｍがさらに好ましく、７０～１５０ｍｍがよりさらに好ましい。第１の挟持片３ａと第２の挟持片３ｂが前記範囲の長さであることにより、肛門から大腸粘膜を直接挟持して採取しやすい。 The length of the first clamping piece 3a and the second clamping piece 3b is preferably 50 to 250 mm, more preferably 100 to 200 mm, still more preferably 70 to 200 mm, and even more preferably 70 to 150 mm. Since the first clamping piece 3a and the second clamping piece 3b have a length within the above range, the colonic mucosa is easily clamped and collected from the anus.
　大腸粘膜を比較的組織の破損が少ない状態で採取するために、第１の挟持面５ａと第２の挟持面５ｂの少なくとも一方はカップ形状であることが好ましい。両者の少なくとも一方がカップ形状の場合であるため、第１の挟持面５ａの辺縁部６ａと第２の挟持面５ｂの辺縁部６ｂが接した場合に、内部に空間が形成される。第１の挟持面５ａと第２の挟持面５ｂに挟持された大腸粘膜のうち、当該空間内に収容された部分は、大腸粘膜を引きちぎる際に負荷があまりかからず、よって組織の破壊を抑制できる。図１に示すように、両方がカップ形状であることにより、大腸粘膜の採取がより容易であり、かつ組織の破壊を抑制できる。 In order to collect the colonic mucosa with relatively little tissue damage, at least one of the first clamping surface 5a and the second clamping surface 5b is preferably cup-shaped. Since at least one of them is a cup shape, when the edge 6a of the first clamping surface 5a and the edge 6b of the second clamping surface 5b are in contact with each other, a space is formed inside. Of the large intestine mucosa sandwiched between the first sandwiching surface 5a and the second sandwiching surface 5b, the portion accommodated in the space does not take much load when the large intestine mucosa is torn, thus destroying the tissue. Can be suppressed. As shown in FIG. 1, since both are cup-shaped, it is easier to collect the mucosa of the large intestine, and tissue destruction can be suppressed.
　第１の挟持面５ａと第２の挟持面５ｂがカップ形状の場合、辺縁部６ａと辺縁部６ｂの内径は、必要な量の大腸粘膜が採取できる大きさに設定すればよい。本発明に係る判定方法に供される大腸粘膜の場合、少量の粘膜が採取できれば十分である。例えば、辺縁部６ａと辺縁部６ｂの内径を、１～５ｍｍ、好ましくは２～３ｍｍとすることにより、大腸粘膜を過度に傷つけることなく、充分量の大腸粘膜を採取することができる。 When the first sandwiching surface 5a and the second sandwiching surface 5b are cup-shaped, the inner diameters of the marginal portion 6a and the marginal portion 6b may be set to such a size that a necessary amount of large intestine mucosa can be collected. In the case of the large intestine mucosa used in the determination method according to the present invention, it is sufficient if a small amount of mucosa can be collected. For example, by setting the inner diameters of the edge portion 6a and the edge portion 6b to 1 to 5 mm, preferably 2 to 3 mm, a sufficient amount of the large intestine mucosa can be collected without excessively damaging the large intestine mucosa.
　辺縁部６ａと辺縁部６ｂは、互いに密接することが可能であればよく、平坦であってもよいが、図１（Ｃ）に示すように、鋸歯状であることが好ましい。鋸歯状の場合には、辺縁部６ａ’と辺縁部６ｂ’により挟持することによって比較的弱い力で大腸粘膜を切断し採取することができる。 The edge portion 6a and the edge portion 6b may be flat as long as they can be brought into close contact with each other, but are preferably serrated as shown in FIG. In the case of a sawtooth shape, the large intestine mucosa can be cut and collected with a relatively weak force by being sandwiched between the edge 6a 'and the edge 6b'.
　第１の挟持片３ａと第２の挟持片３ｂのいずれか一方の内側に突起部８ａを、他方に筒部９ａを、互いに対向するように形成していてもよい。第１の挟持片３ａと第２の挟持片３ｂへ加力し、辺縁部６ａと辺縁部６ｂが互いに接する状態において、突起部８ａの先端は筒部９ａに嵌る。突起部８ａの先端が筒部９ａに嵌っていることにより、採取具２を大腸粘膜から離す際に辺縁部６ａと辺縁部６ｂがずれることなく、安定して大腸粘膜を採取することができる。 The protruding portion 8a may be formed on the inner side of one of the first holding piece 3a and the second holding piece 3b, and the cylindrical portion 9a may be formed on the other side so as to face each other. When the edge 6a and the edge 6b are in contact with each other by applying force to the first holding piece 3a and the second holding piece 3b, the tip of the protrusion 8a fits into the tube 9a. Since the tip of the protrusion 8a is fitted into the tube portion 9a, it is possible to stably collect the large intestine mucosa without the edge 6a and the edge 6b being displaced when the collection tool 2 is separated from the large intestine mucosa. it can.
　図１（Ｄ）は採取具２Ａの変形例である採取具２Ｂの説明図であり、より詳細には、採取具２Ｂの第１の挟持片３ａと第２の挟持片３ｂが加力されていない状態の斜視図である。第１の挟持片３ａは、中心部よりも第１の挟持面５ａが形成されている端部側に、第１の屈曲部７ａを有していてもよい。第２の挟持片３ｂは、中心部よりも第２の挟持面５ｂが形成されている端部側に、第２の屈曲部７ｂを有していてもよい。第１の挟持片３ａと第２の挟持片３ｂが、中心部よりも挟持面が形成されている先端側において、互いに対向している状態を維持したまま、傾斜していることにより、採取補助具１１のスリット１３を貫通して大腸粘膜に接することが容易になる。具体的には、図１（Ｄ）に示すように、第１の挟持片３ａの中心部から連結部４側と第２の挟持片３ｂの中心部から連結部４側とを載せた仮想平面Ｐに交差するように屈曲している。屈曲の角度θ１は、１０～５０°が好ましく、２０～４０°がより好ましく、２５～３５°がさらに好ましい。また、第１の屈曲部７ａから第１の挟持面５ａの先端部までの長さ、及び第２の屈曲部７ｂから第２の挟持面５ｂの先端部までの長さは、２０～６０ｍｍが好ましく、３０～５０ｍｍがより好ましい。屈曲部から挟持面の先端部までの長さが前記範囲内であることにより、採取補助具１１のスリット１３を貫通した状態での粘膜採取がより容易になる。 FIG. 1D is an explanatory diagram of a sampling tool 2B which is a modification of the sampling tool 2A. More specifically, the first clamping piece 3a and the second clamping piece 3b of the sampling tool 2B are applied with force. FIG. The 1st clamping piece 3a may have the 1st bending part 7a in the edge part side in which the 1st clamping surface 5a is formed rather than the center part. The 2nd clamping piece 3b may have the 2nd bending part 7b in the edge part side in which the 2nd clamping surface 5b is formed rather than the center part. The first sandwiching piece 3a and the second sandwiching piece 3b are inclined while maintaining a state in which they are opposed to each other on the distal end side where the sandwiching surface is formed with respect to the center portion, thereby assisting collection. It becomes easy to penetrate the slit 13 of the tool 11 and contact the colonic mucosa. Specifically, as shown in FIG. 1D, a virtual plane in which the connecting portion 4 side from the center of the first holding piece 3a and the connecting portion 4 side from the center of the second holding piece 3b are placed. Bent to intersect P. The bending angle θ 1 is preferably 10 to 50 °, more preferably 20 to 40 °, and even more preferably 25 to 35 °. Further, the length from the first bent portion 7a to the tip portion of the first clamping surface 5a and the length from the second bent portion 7b to the tip portion of the second holding surface 5b are 20 to 60 mm. 30 to 50 mm is more preferable. When the length from the bent portion to the distal end portion of the clamping surface is within the above range, it is easier to collect the mucous membrane in a state where it passes through the slit 13 of the collection assisting tool 11.
　図２（Ａ）～図２（Ｅ）は、採取具２Ａの別の変形例である採取具２Ｃの説明図である。図２（Ａ）は、採取具２の第１の挟持片３ａと第２の挟持片３ｂが加力されていない状態の正面図であり、図２（Ｂ）は、採取具２Ｃの平面図である。図２（Ｃ）は、採取具２Ｃの突起部８ｂの拡大図であり、図２（Ｄ）は、採取具２Ｃの先端部分の、突起部８ｂの係止爪が筒部９ｂの開口縁部の張り出し部に係止されている状態の平面図であり、図２（Ｅ）は、採取具２Ｃの先端部分の、第１の挟持面５ａと第２の挟持面５ｂが接着した状態の平面図である。 2 (A) to 2 (E) are explanatory views of a sampling tool 2C, which is another modification of the sampling tool 2A. FIG. 2 (A) is a front view showing a state in which the first clamping piece 3a and the second clamping piece 3b of the sampling tool 2 are not pressed, and FIG. 2 (B) is a plan view of the sampling tool 2C. It is. FIG. 2C is an enlarged view of the protruding portion 8b of the sampling tool 2C, and FIG. 2D is an opening edge of the cylindrical portion 9b where the locking claw of the protruding portion 8b is at the tip of the sampling tool 2C. FIG. 2E is a plan view showing a state in which the first clamping surface 5a and the second clamping surface 5b are bonded to each other at the distal end portion of the sampling tool 2C. FIG.
　被検者の直腸から粘膜組織を採取する際に、採取具２は、第１の挟持片３ａと第２の挟持片３ｂの距離が開いた状態よりも閉じた状態のほうが、採取補助具１１のスリット１３を貫通させやすい。そこで、図２の突起部８ｂが示すように、採取具２の突起部は、係止爪であってもよい。突起部８ｂの係止爪は、１つ又は２つ以上であってもよく、筒部９ｂの開口縁部の張り出し部に係止できれば幾つであってもよい。この場合には、突起部８ｂを篏合させる筒部９ｂは、開口縁部に径方向内側に張り出し部を設けたものとし、突起部８ｂの係止爪が筒部９ｂの開口縁部の張り出し部に係止させることができる（図２（Ｄ））。突起部８ｂの係止爪の高さは、筒部９ｂの張り出し部に係止させた状態のときに、第１の挟持面５ａと第２の挟持面５ｂの先端部が近接しているが、第１の挟持面５ａと第２の挟持面５ｂが接着していない状態となり、かつ第１の挟持片３ａと第２の挟持片３ｂをさらに加力することによって、突起部８ｂの先端部が筒部９ｂの底部を突き抜けることなく第１の挟持面５ａと第２の挟持面５ｂを接着させることが可能となるように調整することが好ましい。これにより、採取具２の第１の挟持片３ａと第２の挟持片３ｂを加力することなく、第１の挟持面５ａと第２の挟持面５ｂの先端部を近接した状態で安定させることができる。採取具２は、図２（Ｄ）の状態で採取補助具１１のスリット１３を貫通させ、先端部が直腸粘膜組織に接触したら、第１の挟持片３ａと第２の挟持片３ｂを加力し、粘膜組織の一部を挟み込むようにして図２（Ｅ）の状態にし、粘膜組織を採取する。 When collecting mucosal tissue from the rectum of the subject, the collection tool 2 is more in a closed state than in a state in which the distance between the first holding piece 3a and the second holding piece 3b is open. It is easy to penetrate the slit 13. Therefore, as shown by the protrusion 8b in FIG. 2, the protrusion of the collection tool 2 may be a locking claw. There may be one or more locking claws of the protruding portion 8b, and any number may be used as long as it can be locked to the overhanging portion of the opening edge of the cylindrical portion 9b. In this case, the cylindrical portion 9b for mating the protruding portion 8b is provided with a protruding portion on the opening edge radially inward, and the locking claw of the protruding portion 8b protrudes from the opening edge of the cylindrical portion 9b. (Fig. 2D). The height of the locking claw of the protrusion 8b is such that the tip of the first clamping surface 5a and the second clamping surface 5b are close to each other when locked to the overhanging portion of the tube portion 9b. The first sandwiching surface 5a and the second sandwiching surface 5b are not bonded to each other, and the first sandwiching piece 3a and the second sandwiching piece 3b are further applied, whereby the tip of the protruding portion 8b. It is preferable to adjust so that the first clamping surface 5a and the second clamping surface 5b can be bonded without penetrating through the bottom of the cylindrical portion 9b. This stabilizes the front end portions of the first clamping surface 5a and the second clamping surface 5b close to each other without applying force to the first clamping piece 3a and the second clamping piece 3b of the sampling tool 2. be able to. When the collection tool 2 penetrates the slit 13 of the collection assisting tool 11 in the state of FIG. 2D and the tip part contacts the rectal mucosa tissue, the first clamping piece 3a and the second clamping piece 3b are applied with force. Then, a part of the mucosal tissue is sandwiched and the state shown in FIG. 2E is obtained, and the mucosal tissue is collected.
　採取具２は、第１の挟持片３ａと第２の挟持片３ｂに、それぞれ、連結部と屈曲部の間に対応する緩衝部１０ａを設けてもよい。緩衝部１０ａは、その先端に弾性部１０ｂを備えており、突起部８ｂの係止爪が筒部９ｂの開口縁部の張り出し部に係止させた状態では、互いに弾性部１０ｂで接着する（図２（Ｄ））。この緩衝部により、採取具２はより安定的に、突起部８ｂの係止爪が筒部９ｂの開口縁部の張り出し部に係止した状態を維持できる。第１の挟持片３ａと第２の挟持片３ｂをさらに加力した場合にも、先端の弾性部１０ｂが押圧により変形するため、第１の挟持面５ａと第２の挟持面５ｂを接着させることができる（図２（Ｅ））。 The sampling tool 2 may be provided with a buffering portion 10a corresponding to a portion between the connecting portion and the bent portion in the first holding piece 3a and the second holding piece 3b, respectively. The buffer portion 10a is provided with an elastic portion 10b at the tip thereof, and is bonded to each other by the elastic portion 10b in a state where the locking claw of the projection portion 8b is locked to the protruding portion of the opening edge portion of the cylindrical portion 9b ( FIG. 2 (D)). By this buffer part, the sampling tool 2 can maintain the state which the latching claw of the projection part 8b latched to the overhang | projection part of the opening edge part of the cylinder part 9b more stably. Even when the first sandwiching piece 3a and the second sandwiching piece 3b are further applied, the elastic portion 10b at the tip is deformed by pressing, so that the first sandwiching surface 5a and the second sandwiching surface 5b are bonded. (FIG. 2E).
　図３（Ａ）及び図３（Ｂ）は採取補助具１１の一実施態様である採取補助具１１Ａの説明図である。図３（Ａ）は採取補助具１１Ａの下側からみた斜視図であり、図３（Ｂ）は採取補助具１１Ａのスリット側からみた底面図である。図３（Ａ）に示すように、採取補助具１１Ａは、採取具導入部１２と、スリット１３と、把持部１４と、を有する。 3 (A) and 3 (B) are explanatory views of a collection assisting tool 11A which is an embodiment of the collection assisting tool 11. FIG. FIG. 3A is a perspective view seen from the lower side of the collection assisting tool 11A, and FIG. 3B is a bottom view seen from the slit side of the collection assisting tool 11A. As shown in FIG. 3A, the collection assisting tool 11A has a collection tool introducing part 12, a slit 13, and a gripping part 14.
　採取具導入部１２は、側壁にスリット１３を有する円錐台形状の部材である。採取具導入部１２は、外径が小さい先端辺縁部１５から肛門に挿入し、外径が大きい手元辺縁部１６から採取具２を挿入する。採取具導入部１２は、回転軸方向に貫通孔を有していてもよい。肛門への挿入のしやすさの点から、手元辺縁部１６の外径は３０～７０ｍｍが好ましく、４０～５０ｍｍがより好ましい。また、採取具２の大腸粘膜表面への導入しやすさの点から、先端辺縁部１５の外径は１０～３０ｍｍが好ましく、１５～２５ｍｍがより好ましい。同様に、採取具導入部１２の回転軸方向の長さは５０～１５０ｍｍが好ましく、７０～１３０ｍｍがより好ましく、８０～１２０ｍｍがさらに好ましい。 The sampling tool introduction part 12 is a truncated cone-shaped member having a slit 13 on the side wall. The sampling tool introduction unit 12 is inserted into the anus from the distal edge 15 having a small outer diameter, and the sampling tool 2 is inserted from the proximal edge 16 having a large outer diameter. The collection tool introduction part 12 may have a through hole in the rotation axis direction. From the viewpoint of ease of insertion into the anus, the outer diameter of the hand edge 16 is preferably 30 to 70 mm, and more preferably 40 to 50 mm. Further, from the viewpoint of easy introduction of the collection tool 2 into the surface of the large intestine mucosa, the outer diameter of the distal edge portion 15 is preferably 10 to 30 mm, and more preferably 15 to 25 mm. Similarly, the length of the collection tool introduction part 12 in the rotation axis direction is preferably 50 to 150 mm, more preferably 70 to 130 mm, and even more preferably 80 to 120 mm.
　スリット１３は、採取具導入部１２の先端辺縁部１５から手元辺縁部１６に向かって設けられている。採取具導入部１２の側壁の一部分に先端辺縁部１５に達するスリット１３があることにより、腸管内における採取具２の先端部の動きの自由度が高まり、内部構造が複雑な直腸内において、大腸粘膜をより容易に採取することができる。スリット１３は、採取具導入部１２のいずれの位置に設定されていてもよい。例えば、スリット１３は、図３（Ａ）に示すように、把持部１４に近い側にあることが好ましい。また、採取具導入部１２に設けられるスリット１３の数は、１つであってもよく、２以上であってもよい。 The slit 13 is provided from the front edge portion 15 of the sampling tool introduction portion 12 toward the hand edge portion 16. The presence of the slit 13 reaching the distal edge 15 in a part of the side wall of the collection tool introducing portion 12 increases the freedom of movement of the distal end portion of the collection tool 2 in the intestine, and in the rectum having a complicated internal structure, Large intestine mucosa can be collected more easily. The slit 13 may be set at any position of the collection tool introduction unit 12. For example, the slit 13 is preferably on the side close to the gripping portion 14 as shown in FIG. Moreover, the number of the slits 13 provided in the collection tool introduction part 12 may be one, or two or more.
　スリット１３を貫通して採取具２は大腸粘膜表面に達するため、スリット１３の幅は、辺縁部６ａと辺縁部６ｂとが接した状態における、採取具２の第１の挟持面５ａと第２の挟持面５ｂの幅よりも広く設計される。また、スリット１３の幅は、一定であってもよいが、図３（Ｂ）に示すように、先端辺縁部１５から手元辺縁部１６側にいくにつれて広くなっているほうが好ましい。例えば、辺縁部６ａと辺縁部６ｂとが接した状態における、採取具２の第１の挟持面５ａと第２の挟持面５ｂの幅Ｌ１（図１（Ｂ）参照。）が３～８ｍｍの場合、スリット１３の先端辺縁部１５側の幅Ｌ２（図３（Ｂ）参照。）は７～１５ｍｍが好ましく、スリット１３の手元辺縁部１６側の幅Ｌ３（図３（Ｂ）参照。）は１０～２０ｍｍが好ましい。なお、スリット１３は、採取具導入部１２の壁面に２以上形成されていてもよい。 Since the collection tool 2 passes through the slit 13 and reaches the surface of the large intestine mucosa, the width of the slit 13 is the same as that of the first clamping surface 5a of the collection tool 2 in a state where the edge 6a and the edge 6b are in contact with each other. It is designed wider than the width of the second clamping surface 5b. Further, the width of the slit 13 may be constant, but as shown in FIG. 3B, it is preferable that the slit 13 becomes wider from the front edge portion 15 toward the hand edge portion 16 side. For example, the width L 1 (see FIG. 1 (B)) of the first clamping surface 5a and the second clamping surface 5b of the sampling tool 2 in a state where the edge portion 6a and the edge portion 6b are in contact with each other is 3. In the case of ˜8 mm, the width L 2 (see FIG. 3B) of the slit 13 on the front edge portion 15 side is preferably 7 to 15 mm, and the width L 3 of the slit 13 on the hand edge portion 16 side (FIG. 3). (See (B)) is preferably 10 to 20 mm. Note that two or more slits 13 may be formed on the wall surface of the collection tool introduction portion 12.
　把持部１４は、その一端が、採取具導入部１２の手元辺縁部１６近傍に、採取具導入部１２から遠ざかる方向に連結している。把持部１４の長さは、手による握りやすさ等から、５０～１５０ｍｍが好ましく、７０～１３０ｍｍがより好ましい。把持部１４の形状は、握りやすい形状であればどのような形状であってもよく、例えば、板状であってもよく、棒状であってもよく、その他の形状であってもよい。 One end of the gripping part 14 is connected to the vicinity of the hand edge 16 of the collection tool introduction part 12 in a direction away from the collection tool introduction part 12. The length of the gripping portion 14 is preferably 50 to 150 mm, more preferably 70 to 130 mm, from the viewpoint of ease of gripping with a hand. The shape of the gripping portion 14 may be any shape as long as it is easy to grip, and may be, for example, a plate shape, a rod shape, or other shapes.
　図４は、採取補助具１１Ａの変形例である採取補助具１１Ｂの説明図である。図４（Ａ）は採取補助具１１Ｂの上側からみた斜視図であり、図４（Ｂ）は下側からみた斜視図である。また、図４（Ｃ）～図４（Ｇ）は、それぞれ、採取補助具１１Ｂの正面図、平面図、底面図、左側面図、及び右側面図である。採取補助具の把持部は、把持部１４に示すように、下方が開口した中空の棒状であって、リブで補強されているものであってもよい。 FIG. 4 is an explanatory diagram of a collection assisting tool 11B which is a modification of the collection assisting tool 11A. 4A is a perspective view seen from the upper side of the collection assisting tool 11B, and FIG. 4B is a perspective view seen from the lower side. 4 (C) to 4 (G) are a front view, a plan view, a bottom view, a left side view, and a right side view of the collection assisting tool 11B, respectively. As shown in the grip 14, the gripping part of the collection assisting tool may be a hollow rod having an opening at the bottom and reinforced with a rib.
　図５は、本発明に係る大腸粘膜採取用キット１の使用の態様を示す説明図である。まず、大腸粘膜を採取される被検者の肛門に、採取補助具１１を先端辺縁部１５から挿入する。把持部１４を片手で持ち安定させた状態で、手元辺縁部１６側の開口部から採取具２を導入する。導入された採取具２は、先端部からスリット１３を貫通して大腸粘膜表面に達する。採取具２の挟持面５ａと挟持面５ｂとで大腸粘膜を挟持した状態（挟持面５）で、採取具２をスリット１３から引き出すことにより、大腸粘膜が採取できる。 FIG. 5 is an explanatory diagram showing a mode of use of the colonic mucosa collection kit 1 according to the present invention. First, the collection assisting tool 11 is inserted from the distal edge portion 15 into the anus of the subject from whom the colonic mucosa is collected. The sampling tool 2 is introduced from the opening on the side of the hand edge 16 in a state where the grip 14 is held and stabilized with one hand. The introduced collection tool 2 penetrates the slit 13 from the tip and reaches the surface of the large intestine mucosa. The colonic mucosa can be collected by pulling out the sampling tool 2 from the slit 13 while the colonic mucosa is sandwiched between the clamping surface 5a and the clamping surface 5b of the sampling tool 2 (the clamping surface 5).
　次に実施例等を示して本発明をさらに詳細に説明するが、本発明はこれらに限定されるものではない。 Next, the present invention will be described in more detail with reference to examples and the like, but the present invention is not limited thereto.
［実施例１］
　潰瘍性大腸炎患者のうち、内視鏡検査での生検組織による病理診断により大腸癌と診断され、外科手術を施行した患者（ＵＣ癌患者）８名（男性７名、女性１名）と、内科的治療不応潰瘍性大腸炎患者であり、癌以外で外科手術を施行した患者（非癌ＵＣ患者）８名（男性７名、女性１名）とから採取された大腸粘膜中のＤＮＡに対して、ＣｐＧサイトのメチル化率を網羅的に解析した。なお、ＵＣ癌患者８名の平均年齢は４７．１±１２．４歳であり、罹病期間の平均は１１．４±７．３年であった。非癌ＵＣ患者８名の平均年齢は４４．３±１６．４歳であり、罹病期間の平均は６．５±５．２年であった。 [Example 1]
Among the patients with ulcerative colitis, 8 patients (7 men, 1 woman) who were diagnosed with colon cancer by pathological diagnosis by biopsy tissue in endoscopy and who underwent surgery (7 men, 1 woman) , DNA in colonic mucosa collected from 8 patients (7 men, 1 woman) who have undergone surgery other than cancer (non-cancer UC patients) who are refractory to medical treatment ulcerative colitis In contrast, the methylation rate of the CpG site was comprehensively analyzed. The average age of 8 UC cancer patients was 47.1 ± 12.4 years, and the average disease duration was 11.4 ± 7.3 years. The average age of the 8 non-cancer UC patients was 44.3 ± 16.4 years, and the average disease duration was 6.5 ± 5.2 years.
＜ＣｐＧサイトのメチル化レベルの網羅的解析＞
（１）生検及びＤＮＡ抽出
　同一患者の大腸３か所から粘膜組織を採取し、常法に従いホルマリン固定パラフィン包埋（ＦＦＰＥ）サンプルを作製した。採取部位は、ＵＣ癌患者は盲腸、直腸、及び癌部とし、非癌ＵＣ患者は盲腸、横行結腸、直腸とした。ＦＦＰＥサンプルから切片を切り出し、QIAmp DNA FFPE tissue kit（Qiagen社製）を使用してＤＮＡを抽出した。 <Comprehensive analysis of methylation level of CpG site>
(1) Biopsy and DNA extraction Mucosal tissues were collected from three large intestines of the same patient, and formalin-fixed paraffin-embedded (FFPE) samples were prepared according to a conventional method. The collection sites were the cecum, rectum, and cancer for UC cancer patients, and the cecum, transverse colon, and rectum for non-cancer UC patients. A section was cut out from the FFPE sample, and DNA was extracted using a QIAmp DNA FFPE tissue kit (manufactured by Qiagen).
（２）ＤＮＡサンプルの品質評価
　得られたＤＮＡの濃度は次のようにして求めた。すなわち、Quant-iT PicoGreen dsDNA Assay Kit（Life Technologies社製）を用いて各サンプルの蛍光強度を測定し、キット付属のλ－ＤＮＡの検量線を用いて濃度を算出した。
　次に、各サンプルをＴＥ（ｐＨ８．０）にて１ｎｇ／μＬに希釈し、Illumina FFPE QC Kit（Illumina社製）及びFast SYBR Green Master Mix（Life Technologies社製）を用いてリアルタイムＰＣＲを行い、Ｃｔ値を求めた。サンプルとポジティブコントロールとのＣｔ値の差（以下、ΔＣｔ値）をサンプルごとに算出し、品質を評価した。ΔＣｔ値が５未満のサンプルは品質良好と判断し、以降のステップに進めた。 (2) Quality evaluation of DNA sample The concentration of the obtained DNA was determined as follows. Specifically, the fluorescence intensity of each sample was measured using Quant-iT PicoGreen dsDNA Assay Kit (manufactured by Life Technologies), and the concentration was calculated using the λ-DNA calibration curve attached to the kit.
Next, each sample was diluted to 1 ng / μL with TE (pH 8.0), and real-time PCR was performed using Illumina FFPE QC Kit (manufactured by Illumina) and Fast SYBR Green Master Mix (manufactured by Life Technologies). Ct value was determined. The difference in Ct value between the sample and the positive control (hereinafter referred to as ΔCt value) was calculated for each sample, and the quality was evaluated. Samples with a ΔCt value of less than 5 were judged to have good quality and proceeded to the subsequent steps.
（３）バイサルファイト処理
　EZ DNA Methylation Kit（ZYMO RESEARCH社製）を使用して、ＤＮＡサンプルに対してバイサルファイト処理を実施した。 (3) Bisulfite treatment Bisulfite treatment was performed on DNA samples using EZ DNA Methylation Kit (manufactured by ZYMO RESEARCH).
（４）分解ＤＮＡの修復及び全ゲノム増幅
　バイサルファイト処理後のＤＮＡに対して、Infinium HD FFPE Restore Kit（Illumina社製）を使用し、分解ＤＮＡを修復した。修復されたＤＮＡをアルカリ変性した後、中和させ、HumanMethylation450 DNA Analysis Kit（Illumina社製）の全ゲノム増幅用の酵素とプライマーを添加し、３７℃のIncubation Oven（Illumina社製）で２０時間以上、等温で反応させることによって、全ゲノムを増幅させた。 (4) Repair of degraded DNA and amplification of whole genome Degraded DNA was repaired by using Infinium HD FFPE Restore Kit (manufactured by Illumina) for the DNA after bisulfite treatment. The repaired DNA is alkali-denatured, neutralized, added with the enzyme and primers for whole genome amplification of HumanMethylation450 DNA Analysis Kit (Illumina), and incubated for more than 20 hours at 37 ° C Incubation Oven (Illumina) The whole genome was amplified by reacting isothermally.
　（５）全ゲノム増幅したＤＮＡの断片化と精製
　全ゲノム増幅したＤＮＡに、HumanMethylation450 DNA Analysis Kit（Illumina社製）の断片化用の酵素を添加し、Microsample Incubator（SciGene）で３７℃、１時間反応させた。断片化したＤＮＡに、共沈剤と２－プロパノールを加えて遠心分離処理し、ＤＮＡを沈澱させた。 (5) Fragmentation and purification of whole-genome amplified DNA To the whole-genome amplified DNA, the fragmentation enzyme of HumanMethylation450 DNA Analysis Kit (manufactured by Illumina) was added, and 37 ° C for 1 hour with Microsample Incubator (SciGene) Reacted. To the fragmented DNA, a coprecipitation agent and 2-propanol were added and centrifuged to precipitate the DNA.
（６）ハイブリダイゼーション
　沈澱させたＤＮＡに、Hybridization bufferを加え、４８℃のHybridization Oven（Illumina社製）で１時間反応させ、ＤＮＡを溶解させた。溶解させたＤＮＡを９５℃のMicrosample Incubator（SciGene社製）で２０分間インキュベートして１本鎖に変性させた後、HumanMethylation450 DNA Analysis Kit（Illumina社製）のBeadChip上に分注した。４８℃のHybridization Ovenで１６時間以上反応させ、BeadChip上のプローブと１本鎖ＤＮＡをハイブリダイズした。 (6) Hybridization Hybridization buffer was added to the precipitated DNA, and the mixture was reacted with Hybridization Oven (manufactured by Illumina) at 48 ° C. for 1 hour to dissolve the DNA. The dissolved DNA was incubated with a 95 ° C. Microsample Incubator (manufactured by SciGene) for 20 minutes to denature into single strands, and then dispensed onto a BeadChip of a HumanMethylation450 DNA Analysis Kit (manufactured by Illumina). The reaction was carried out for 16 hours or more in Hybridization Oven at 48 ° C., and the probe on the BeadChip and the single-stranded DNA were hybridized.
（７）標識反応及びスキャニング
　ハイブリダイゼーション後のBeadChip上のプローブを伸長反応させ、蛍光色素を結合させた。次いで、当該BeadChipをiSCANシステム（Illumina社製）でスキャンし、メチル化蛍光強度及び非メチル化蛍光強度を測定した。実験終了時に、スキャンデータが全て揃っていること、及びスキャンが正常に行われたことを確認した。 (7) Labeling reaction and scanning The probe on the BeadChip after hybridization was subjected to an extension reaction to bind a fluorescent dye. Subsequently, the BeadChip was scanned with an iSCAN system (manufactured by Illumina), and methylated fluorescence intensity and unmethylated fluorescence intensity were measured. At the end of the experiment, it was confirmed that all the scan data was available and that the scan was performed normally.
（８）ＤＮＡメチル化レベルの定量及び比較解析
　ＤＮＡメチル化解析ソフトウェアGenomeStudio（Version：V2011.1）を用いてスキャンデータを解析した。ＤＮＡメチル化レベル（β値）は、次の式により算出した。 (8) Quantification of DNA methylation level and comparative analysis Scan data was analyzed using DNA methylation analysis software GenomeStudio (Version: V2011.1). The DNA methylation level (β value) was calculated by the following formula.
［β値］＝
　［メチル化蛍光強度］÷ (［メチル化蛍光強度］＋［非メチル化蛍光強度］＋100) [Β value] =
[Methylated fluorescence intensity] ÷ ([Methylated fluorescence intensity] + [Unmethylated fluorescence intensity] +100)
　メチル化レベルが高い場合、β値は１に近づき、メチル化レベルが低い場合、β値は０に近づく。非癌ＵＣ患者直腸サンプル群（ｎ＝８）に対するＵＣ癌患者直腸サンプル群（ｎ＝８）のＤＮＡメチル化レベルの比較解析には、GenomeStudioにより算出されるDiffScoreを用いた。両者のＤＮＡメチル化レベルが近い場合、DiffScoreは０に近づき、ＵＣ癌患者でのレベルが高い場合は正の値を示し、低い場合は負の値を示す。両群のメチル化レベルの差異が大きいほど、DiffScoreの絶対値は大きくなる。また、ＵＣ癌患者直腸サンプル群（ｎ＝８）の平均β値から非癌ＵＣ患者直腸サンプル群（ｎ＝８）の平均β値を差し引いた値（Δβ値）も比較解析に用いた。 When the methylation level is high, the β value approaches 1 and when the methylation level is low, the β value approaches 0. DiffScore calculated by GenomeStudio was used for comparative analysis of the DNA methylation level of the UC cancer patient rectal sample group (n = 8) to the non-cancer UC patient rectal sample group (n = 8). When both DNA methylation levels are close, DiffScore approaches 0, showing a positive value when the level is high in UC cancer patients, and a negative value when low. The greater the difference in methylation level between the two groups, the greater the absolute value of DiffScore. In addition, a value (Δβ value) obtained by subtracting the average β value of the non-cancer UC patient rectal sample group (n = 8) from the average β value of the UC cancer patient rectal sample group (n = 8) was also used for the comparative analysis.
　なお、ＤＮＡメチル化定量とＤＮＡメチル化レベル比較解析には、GenomeStudioとソフトウェアMethylation Module（Version:1.9.0）を用いた。GenomeStudioの設定条件は、下記の通りである。 In addition, GenomeStudio and software Methylation Module (Version: 1.9.0) were used for DNA methylation quantification and DNA methylation level comparison analysis. The setting conditions of GenomeStudio are as follows.
ＤＮＡメチル化定量；
　Normalization：　有り（Ｃｏｎｔｒｏｌｓ）
　Subtract Background：　有り
　Content Descriptor：　　　 HumanMethylation450_15017482_v.1.2.bpm DNA methylation quantification;
Normalization: Yes (Controls)
Subtract Background: Yes Content Descriptor: HumanMethylation450_15017482_v.1.2.bpm
ＤＮＡメチル化レベル比較解析；
　Normalization：　有り(Controls)
　Subtract Background：　有り
　Content Descriptor：　HumanMethylation450_15017482_v.1.2.bpm
　Ref Group：　比較解析4. Group-3
　Error Model：　Illumina custom
　Compute False Discovery Rate：　無し DNA methylation level comparative analysis;
Normalization: Yes (Controls)
Subtract Background: Yes Content Descriptor: HumanMethylation450_15017482_v.1.2.bpm
Ref Group: Comparative analysis 4. Group-3
Error Model: Illumina custom
Compute False Discovery Rate: None
（９）多変量解析
　ＤＮＡメチル化レベルの定量及び比較解析の結果を用いて、統計解析ソフトウェアＲ（Version: 3.0.1、64bit、Windows（登録商標））を用いて、DiffScoreの算出、クラスター解析及び主成分分析を実施した。 (9) Multivariate analysis Using the results of quantitative and comparative analysis of DNA methylation level, statistical analysis software R (Version: 3.0.1, 64bit, Windows (registered trademark)), DiffScore calculation, cluster analysis And principal component analysis.
クラスター解析のＲスクリプト：
> data.dist<-as.dist(1-cor(data.frame,use="pairwise.complete.obs",method="p"))> hclust(data.dist,method="complete")
　　　 # data.frame: CpG（行）x サンプル（列）から成るデータフレーム
　　　# 1-ピアソン相関係数を距離として定義し、complete linkage法により実施 R script for cluster analysis:
> data.dist <-as.dist (1-cor (data.frame, use = "pairwise.complete.obs", method = "p"))> hclust (data.dist, method = "complete")
# data.frame: Data frame consisting of CpG (rows) x samples (columns) # 1-Pearson correlation coefficient is defined as a distance and implemented by the complete linkage method
主成分分析のＲスクリプト：
> prcomp(t(data.frame),scale=T)
# data.frame: CpG（行）x サンプル（列）から成るデータフレーム R script for principal component analysis:
> prcomp (t (data.frame), scale = T)
# data.frame: data frame consisting of CpG (row) x sample (column)
＜ＣｐＧバイオマーカーの選択＞
（１）ＣｐＧバイオマーカー候補の抽出
　網羅的ＤＮＡメチル化解析データからＧｐＧバイオマーカー候補を選定する手段として、DiffScore及びΔβ値に基づいた絞り込みが報告されている（BMC Med genomics vol.4, p.50, 2011; Sex Dev vol.5, p.70, 2011）。前者はDiffScoreの絶対値が３０超、及びΔβ値の絶対値が０．２超に設定し、後者はDiffScoreの絶対値が３０超、及びΔβ値の絶対値が０．３超に設定して、バイオマーカー候補を抽出している。これらの方法に準じて、BeadChipに搭載されている４８５，５７７個のＣｐＧサイトからバイオマーカー候補を抽出した。 <Selection of CpG biomarker>
(1) Extraction of CpG biomarker candidates As a means for selecting GpG biomarker candidates from comprehensive DNA methylation analysis data, narrowing based on DiffScore and Δβ values has been reported (BMC Med genomics vol.4, p. 50, 2011; Sex Dev vol.5, p.70, 2011). The former sets the absolute value of DiffScore to more than 30 and the absolute value of Δβ value to more than 0.2, and the latter sets the absolute value of DiffScore to more than 30 and the absolute value of Δβ value to more than 0.3. Biomarker candidates are extracted. In accordance with these methods, biomarker candidates were extracted from 485,577 CpG sites mounted on BeadChip.
　具体的には、まず、４８５，５７７個のＣｐＧサイトから、DiffScoreの絶対値が３０超である７２，９０５個のＣｐＧサイトを選択した。BeadChipには、特許文献１に記載されているｍｉＲ－１、ｍｉＲ－９、ｍｉＲ－１２４、ｍｉＲ－１３７、ｍｉＲ－３４ｂ／ｃの各遺伝子領域に位置する８６個のＣｐＧサイトも搭載されていたが、このうち、DiffScoreの絶対値が３０超であったのは２７個であった。
　次に、この７２，９０５個のＣｐＧサイトの中から、Δβ値の絶対値が０．３超である３２個のＣｐＧサイトを抽出した。以下、この３２個のＣｐＧサイトをまとめて「３２ＣｐＧセット」という。この時点で、特許文献１に記載されている各遺伝子領域に位置するＣｐＧサイトは、全て除外されてしまった。
　さらに、癌患者を取りこぼしなく判別することを目的に、癌患者サンプル内でＤＮＡメチル化レベルの変動が少ないものを絞り込んだ。すなわち、ＵＣ癌患者２４サンプル（３部位×各部位８サンプル）のβ値の不偏分散ｖａｒを求め、不偏分散ｖａｒの値が０．０５より小さい１６個のＣｐＧサイトを選抜した。以下、この１６個のＣｐＧサイトをまとめて「１６ＣｐＧセット」という。１６ＣｐＧセットからさらに、不偏分散ｖａｒの値が０．０３より小さい９個のＣｐＧサイトを絞り込んだ。以下、この９個のＣｐＧサイトをまとめて「９ＣｐＧセット」という。 Specifically, first, 72,905 CpG sites having an absolute DiffScore value of more than 30 were selected from 485,577 CpG sites. BeadChip also had 86 CpG sites located in each gene region of miR-1, miR-9, miR-124, miR-137, miR-34b / c described in Patent Document 1. However, of these, the absolute value of DiffScore was more than 30 and 27.
Next, 32 CpG sites having an absolute value of Δβ value exceeding 0.3 were extracted from the 72,905 CpG sites. Hereinafter, the 32 CpG sites are collectively referred to as a “32 CpG set”. At this point, all CpG sites located in each gene region described in Patent Document 1 have been excluded.
Furthermore, in order to discriminate cancer patients without missing, the cancer patient samples were narrowed down to those with little fluctuation in DNA methylation level. That is, the unbiased variance var of the β value of 24 samples of UC cancer patients (3 sites × 8 samples of each site) was obtained, and 16 CpG sites having an unbiased variance var value smaller than 0.05 were selected. Hereinafter, the 16 CpG sites are collectively referred to as a “16 CpG set”. Nine CpG sites with an unbiased variance var value smaller than 0.03 were further narrowed down from the 16 CpG set. Hereinafter, these nine CpG sites are collectively referred to as a “9CpG set”.
　３２ＣｐＧセットの各ＣｐＧサイトの結果を表１７に示す。表中、「１６ＣｐＧ」欄に＃があるＣｐＧサイトは１６ＣｐＧセットに含まれるものを、「９ＣｐＧ」欄に＃があるＣｐＧサイトは９ＣｐＧセットに含まれるものを、それぞれ示す。 Table 17 shows the results of each CpG site of the 32CpG set. In the table, CpG sites with # in the “16CpG” column indicate those included in the 16CpG set, and CpG sites with # in the “9CpG” column indicate those included in the 9CpG set.
Figure JPOXMLDOC01-appb-T000021
Figure JPOXMLDOC01-appb-T000021
（２）ＣｐＧバイオマーカー候補を用いた臨床サンプルの多変量解析
　前記３２ＣｐＧセット、１６ＣｐＧセット、９ＣｐＧセットを用いて、全４８サンプルのクラスター解析及び主成分分析を行ったところ、図６Ａ、６Ｃ、及び６Ｅに示すように、いずれのＣｐＧセットでも、クラスター解析では、全てのＵＣ癌患者サンプルが同一クラスター（図中、枠内）に集積した。また、図６Ｂ、６Ｄ、及び６Ｆに示すように、主成分分析（縦軸は第２主成分）では、ＵＣ癌患者サンプル（●）と非癌ＵＣ患者サンプル（▲）が第１主成分（横軸）方向にそれぞれ独立のクラスターを形成した。すなわち、いずれのＣｐＧセットでも、ＵＣ癌患者２４サンプルと非癌ＵＣ患者２４サンプルを明確に区別することができた。一方、特許文献１に記載されている各遺伝子領域に位置するＣｐＧサイトから選抜された２７個のＣｐＧサイトを用いて、同様にクラスター解析及び主成分分析を行ったところ、図７Ａ及び７Ｂに示すように、ＵＣ癌患者サンプルと非癌ＵＣ患者サンプルを明確に区別することは出来なかった。これらの結果から、表１７に記載の３２ＣｐＧは、潰瘍性大腸炎患者における大腸癌の発症のバイオマーカーとして極めて有用であり、これらを用いることにより、潰瘍性大腸炎患者の大腸癌の発症の有無を高い感度と特異度によって判定できることが明らかである。 (2) Multivariate analysis of clinical samples using CpG biomarker candidates Cluster analysis and principal component analysis of all 48 samples were performed using the 32CpG set, 16CpG set, and 9CpG set. As shown in 6E, in any CpG set, in the cluster analysis, all UC cancer patient samples were accumulated in the same cluster (in the frame in the figure). As shown in FIGS. 6B, 6D, and 6F, in the principal component analysis (vertical axis is the second principal component), the UC cancer patient sample (●) and the non-cancer UC patient sample (▲) are the first principal component ( Independent clusters were formed in the horizontal axis direction. That is, in any CpG set, 24 UC cancer patient samples and 24 non-cancer UC patient samples could be clearly distinguished. On the other hand, the cluster analysis and the principal component analysis were similarly performed using 27 CpG sites selected from the CpG sites located in each gene region described in Patent Document 1, and the results are shown in FIGS. 7A and 7B. Thus, a UC cancer patient sample and a non-cancer UC patient sample could not be clearly distinguished. From these results, 32CpG described in Table 17 is extremely useful as a biomarker of the onset of colon cancer in patients with ulcerative colitis, and by using these, the presence or absence of onset of colon cancer in patients with ulcerative colitis It is clear that can be determined by high sensitivity and specificity.
　［実施例２］
　実施例１の潰瘍性大腸炎患者とは別に、内視鏡検査での生検組織による病理診断により大腸癌と診断され、外科手術を施行した患者（ＵＣ癌患者）２４名と、内科的治療不応潰瘍性大腸炎患者であり、癌以外で外科手術を施行した患者（非癌ＵＣ患者）２４名とから採取された大腸粘膜中のＤＮＡに対して、ＣｐＧサイトのメチル化率を網羅的に解析した。 [Example 2]
In addition to the patients with ulcerative colitis of Example 1, 24 patients (UC cancer patients) who were diagnosed with colorectal cancer by pathological diagnosis with biopsy tissue by endoscopy and who underwent surgery, and medical treatment Comprehensive CpG site methylation rate for DNA in colon mucosa collected from 24 patients with refractory ulcerative colitis who have undergone surgery other than cancer (non-cancer UC patients) Was analyzed.
　ＣｐＧサイトのメチル化率の解析に供したＤＮＡは、潰瘍性大腸炎患者の直腸の粘膜組織から採取したＦＦＰＥサンプルから、実施例１と同様にしてＤＮＡを抽出し、全ゲノム増幅し、ＣｐＧサイトのＤＮＡメチル化レベルの定量及び比較解析を行い、その結果を用いてDiffScoreの算出、クラスター解析及び主成分分析を実施した。 The DNA used for the analysis of the methylation rate of the CpG site was extracted from the FFPE sample collected from the rectal mucosal tissue of a patient with ulcerative colitis in the same manner as in Example 1, and the whole genome was amplified. Quantification and comparative analysis of DNA methylation levels were performed, and DiffScore calculation, cluster analysis, and principal component analysis were performed using the results.
　（１）ＣｐＧバイオマーカー候補の抽出
　次いで、網羅的ＤＮＡメチル化解析データからＣｐＧバイオマーカー候補を抽出した。　具体的には、まず、４８５，５７７個のＣｐＧサイトから、Δβ値の絶対値が０．２超である３２４個のＣｐＧサイトを抽出した。 (1) Extraction of CpG biomarker candidates Next, CpG biomarker candidates were extracted from comprehensive DNA methylation analysis data. Specifically, first, 324 CpG sites having an absolute value of Δβ value exceeding 0.2 were extracted from 485,577 CpG sites.
　次に、下記の２種類のロジスティック回帰モデルを作成した。
（１）３２４個のＣｐＧサイトから２群のｔ－ｔｅｓｔ検定で上位１００個のＣｐＧサイトを選択し、１００個のＣｐＧから選んだ３個のＣｐＧ全ての組み合わせに基づく１６１，７００個のロジスティック回帰モデル。（２）３２４個のＣｐＧサイトから選んだ２個のＣｐＧの全ての組み合わせに基づく、５２，３２６個のロジスティック回帰モデル。 Next, the following two types of logistic regression models were created.
(1) Top 100 CpG sites were selected from 324 CpG sites by 2 groups of t-test tests, and 161,700 logistic regressions based on combinations of all 3 CpGs selected from 100 CpGs model. (2) 52,326 logistic regression models based on all combinations of two CpGs selected from 324 CpG sites.
　両ロジスティック回帰モデルの判別式について、下記の４つの基準について、それぞれ充足するＣｐＧサイトを選定し、出現するＣｐＧサイトの頻度を算出した。
［基準１］感度が９０％超、特異度が９０％超、かつ判別式の係数ｐ値が０．０５未満。［基準２］感度が９０％超、特異度が９０％超、判別式の係数ｐ値が０．０５未満、かつＡＩＣ（赤池の情報量基準）が３０未満。
［基準３］感度が９５％超、特異度が８５％超、かつ判別式の係数ｐ値が０．０５未満。［基準４］感度が９５％超、特異度が８５％超、判別式の係数ｐ値が０．０５未満、かつＡＩＣが３０未満。 Regarding the discriminants of both logistic regression models, CpG sites that satisfy each of the following four criteria were selected, and the frequency of appearing CpG sites was calculated.
[Standard 1] Sensitivity is over 90%, specificity is over 90%, and coefficient p value of discriminant is less than 0.05. [Standard 2] Sensitivity is more than 90%, specificity is more than 90%, discriminant coefficient p-value is less than 0.05, and AIC (Akaike's information criterion) is less than 30.
[Standard 3] Sensitivity is over 95%, specificity is over 85%, and coefficient p value of discriminant is less than 0.05. [Standard 4] Sensitivity is over 95%, specificity is over 85%, discriminant coefficient p-value is less than 0.05, and AIC is less than 30.
　４つの基準のそれぞれについて、上位１０ＣｐＧサイトを選択したところ、表８～１０に記載の３４個のＣｐＧサイト（３４ＣｐＧセット）が選抜された。各ＣｐＧサイトの結果を表１８に示す。 When the top 10 CpG sites were selected for each of the four criteria, 34 CpG sites (34 CpG sets) listed in Tables 8 to 10 were selected. The results for each CpG site are shown in Table 18.
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-T000022
（２）ＣｐＧバイオマーカー候補を用いた臨床サンプルの多変量解析
　前記３４ＣｐＧセットのメチル化レベルに基づき、全４８サンプルのクラスター解析及び主成分分析を行った。この結果、クラスター解析（図８Ａ）では、大多数のＵＣ癌患者サンプルが同一クラスター（図中、枠内）に集積した。また、主成分分析（図８Ｂ、縦軸は第２主成分）では、ＵＣ癌患者サンプル（●）と非癌ＵＣ患者サンプル（▲）が第１主成分（横軸）方向にそれぞれ独立のクラスターを形成した。すなわち、いずれのＣｐＧセットでも、ＵＣ癌患者２４サンプルと非癌ＵＣ患者２４サンプルを明確に区別することができた。 (2) Multivariate analysis of clinical samples using CpG biomarker candidates Based on the methylation level of the 34CpG set, cluster analysis and principal component analysis of all 48 samples were performed. As a result, in the cluster analysis (FIG. 8A), the majority of UC cancer patient samples accumulated in the same cluster (in the frame in the figure). In the principal component analysis (FIG. 8B, the vertical axis is the second principal component), the UC cancer patient sample (●) and the non-cancer UC patient sample (▲) are independent clusters in the first principal component (horizontal axis) direction. Formed. That is, in any CpG set, 24 UC cancer patient samples and 24 non-cancer UC patient samples could be clearly distinguished.
（３）ＣｐＧバイオマーカー候補を用いた臨床サンプルの大腸癌発症可能性の評価
　前記３４ＣｐＧセットのうち、配列番号３４で表される塩基配列中のＣｐＧサイト（ｃｇ１０９３１１９０）、配列番号３７で表される塩基配列中のＣｐＧサイト（ｃｇ１３６７７１４９）、及び配列番号５６で表される塩基配列中のＣｐＧサイト（ｃｇ１４５１６１００）の３個のＣｐＧサイトのメチル化率をマーカーとした場合の、潰瘍性大腸炎患者の大腸癌発症の有無の判定の精度を調べた。 (3) Evaluation of the likelihood of developing colon cancer in a clinical sample using a CpG biomarker candidate Among the 34 CpG sets, a CpG site (cg10931190) in the base sequence represented by SEQ ID NO: 34, represented by SEQ ID NO: 37 When the methylation rate of 3 CpG sites in the CpG site (cg136767149) in the nucleotide sequence and the CpG site (cg14516100) in the nucleotide sequence represented by SEQ ID NO: 56 is used as a marker, The accuracy of the determination of the presence or absence of colorectal cancer was examined.
　具体的には、２４名のＵＣ癌患者及び２４名の非癌ＵＣ患者の直腸から採取された検体の前記３つのＣｐＧサイトのメチル化レベルの数値（β値）を用いてロジスティック回帰モデルに基づく判別式を作成してＵＣ癌患者と非癌ＵＣ患者の判別を行った。この結果、感度（ＵＣ癌患者のうち、陽性と評価された患者の割合）が９５．８％、特異度（非癌ＵＣ患者のうち、陰性と評価された患者の割合）が９１．７％、陽性的中率（陽性と評価された患者のうち、ＵＣ癌患者の割合）が９２％、陰性的中率（陰性と評価された患者のうち、非癌ＵＣ患者の割合）が９５．６％であり、いずれも９０％以上と高かった。また、図９にＲＯＣ（Receiver Operating Characteristic）曲線を示す。ＡＵＣ（ＲＯＣ曲線下面積）は０．９８であった。これらの結果から、前記３４ＣｐＧセットから選択された数個のＣｐＧサイトのメチル化率に基づいて、高感度かつ高特異度で、潰瘍性大腸炎患者の大腸癌発症の可能性を評価できることが確認された。 Specifically, based on a logistic regression model using numerical values (β values) of methylation levels of the three CpG sites of specimens collected from rectums of 24 UC cancer patients and 24 non-cancer UC patients. A discriminant was created to discriminate between UC cancer patients and non-cancer UC patients. As a result, the sensitivity (the ratio of patients who were evaluated as positive among UC cancer patients) was 95.8%, and the specificity (the ratio of patients who were evaluated as negative among non-cancer UC patients) was 91.7%. The positive predictive value (the ratio of UC cancer patients out of patients evaluated as positive) was 92%, and the negative predictive value (the ratio of non-cancer UC patients out of patients evaluated as negative) was 95.6. %, Both of which were as high as 90% or more. FIG. 9 shows a ROC (Receiver-Operating-Characteristic) curve. The AUC (area under the ROC curve) was 0.98. Based on these results, it was confirmed that the possibility of developing colon cancer in ulcerative colitis patients can be evaluated with high sensitivity and specificity based on the methylation rate of several CpG sites selected from the 34CpG set. It was done.
　［実施例３］
　実施例１において求めた潰瘍性大腸炎患者の直腸から採取した検体の各ＣｐＧサイトのＤＮＡメチル化レベル（β値）と、実施例２において求めた潰瘍性大腸炎患者の各ＣｐＧサイトのＤＮＡメチル化レベル（β値）から、ＣｐＧバイオマーカー候補を抽出した。 [Example 3]
The DNA methylation level (β value) of each CpG site of the specimen collected from the rectum of the ulcerative colitis patient obtained in Example 1 and the DNA methylation of each CpG site of the ulcerative colitis patient obtained in Example 2 CpG biomarker candidates were extracted from the conversion level (β value).
（１）ＣｐＧバイオマーカー候補の抽出
　具体的には、まず、４８５，５７７個のＣｐＧサイトから、Δβ値の絶対値が０．２超である１７２個のＣｐＧサイトを抽出した。次いで、この１７２個のＣｐＧサイトから、実施例２と同様にして、２種類のロジスティック回帰モデルを作成し、前記４つの基準のそれぞれについて、上位１０ＣｐＧサイトを選択した。この結果、表１１及び表１２に記載の１８個のＣｐＧサイト（１８ＣｐＧセット）が選抜された。各ＣｐＧサイトの結果を表１９に示す。 (1) Extraction of CpG biomarker candidates Specifically, 172 CpG sites having an absolute value of Δβ value exceeding 0.2 were first extracted from 485,577 CpG sites. Next, two types of logistic regression models were created from the 172 CpG sites in the same manner as in Example 2, and the top 10 CpG sites were selected for each of the four criteria. As a result, 18 CpG sites (18 CpG set) described in Table 11 and Table 12 were selected. The results for each CpG site are shown in Table 19.
Figure JPOXMLDOC01-appb-T000023
Figure JPOXMLDOC01-appb-T000023
（２）ＣｐＧバイオマーカー候補を用いた臨床サンプルの多変量解析
　前記１８ＣｐＧセットのメチル化レベルに基づき、全６４サンプルのクラスター解析及び主成分分析を行った。この結果、クラスター解析（図１０Ａ）では、大多数のＵＣ癌患者サンプルが同一クラスター（図中、枠内）に集積した。また、主成分分析（図１０Ｂ、縦軸は第２主成分）では、ＵＣ癌患者サンプル（●）と非癌ＵＣ患者サンプル（▲）が第１主成分（横軸）方向にそれぞれ独立のクラスターを形成した。すなわち、いずれのＣｐＧセットでも、ＵＣ癌患者３２サンプルと非癌ＵＣ患者３２サンプルを明確に区別することができた。 (2) Multivariate analysis of clinical samples using CpG biomarker candidates Based on the methylation level of the 18CpG set, cluster analysis and principal component analysis of all 64 samples were performed. As a result, in the cluster analysis (FIG. 10A), the majority of UC cancer patient samples were accumulated in the same cluster (in the frame in the figure). In the principal component analysis (FIG. 10B, the vertical axis is the second principal component), the UC cancer patient sample (●) and the non-cancer UC patient sample (▲) are independent clusters in the first principal component (horizontal axis) direction. Formed. That is, in any CpG set, 32 samples of UC cancer patients and 32 samples of non-cancer UC patients could be clearly distinguished.
［実施例４］
　実施例２において取得したＵＣ癌患者２４名及び非癌ＵＣ患者２４名の直腸から採取した検体の各ＤＭＲの平均メチル化率（平均β値；各ＤＭＲ中に存在するＣｐＧサイトのメチル化レベル（β値）の相加平均値）から、ＤＭＲバイオマーカー候補を抽出した。 [Example 4]
The average methylation rate of each DMR (average β value; methylation level of CpG sites present in each DMR) of specimens collected from the rectums of 24 UC cancer patients and 24 non-cancer UC patients obtained in Example 2 ( DMR biomarker candidates were extracted from the arithmetic average value of β value).
（１）ＤＭＲバイオマーカー候補の抽出
　具体的には、まず、４８５，５７７個のＣｐＧサイトのメチル化データ（ＩＤＡＴ形式）を、ＣｈＡＭＰパイプライン（Bioinformatics、30、428、2014；http://bioconductor.org/packages/release/bioc/html/ChAMP.html）に入力し、ＵＣ癌患者と非癌ＵＣ患者の２群間で有意と判定されたＤＭＲ２，５４９か所を抽出した。この中でΔβ値（［平均β値（ＵＣ癌）］－［平均β値（非癌ＵＣ）］）の絶対値を０．１５超に設定したところ、３９か所に絞り込まれた。さらにΔβ値の絶対値が０．１超である４８４か所のうち、実施例１で取得したＵＣ癌患者と非癌ＵＣ患者とのΔβ値が０．１５超である８０か所を追加し、総数１１２か所（ＤＭＲ番号１～１１２）をＤＭＲバイオマーカー候補とした。この１１２か所のＤＭＲ（１１２ＤＭＲセット）の結果を表２０～２２に示す。 (1) Extraction of DMR biomarker candidates Specifically, first, methylation data (IDAT format) of 485,577 CpG sites was converted into ChAMP pipeline (Bioinformatics, 30, 428, 2014; http: // bioconductor org / packages / release / bioc / html / ChAMP.html), and extracted 2,549 DMRs judged to be significant between the two groups of UC cancer patients and non-cancer UC patients. Among them, when the absolute value of Δβ value ([average β value (UC cancer)] − [average β value (non-cancer UC)]) was set to more than 0.15, it was narrowed down to 39 locations. Furthermore, among the 484 places where the absolute value of Δβ value is more than 0.1, 80 places where the Δβ value between the UC cancer patient and the non-cancer UC patient obtained in Example 1 is more than 0.15 are added. A total of 112 locations (DMR numbers 1-112) were designated as DMR biomarker candidates. The results of these 112 DMRs (112 DMR sets) are shown in Tables 20-22.
Figure JPOXMLDOC01-appb-T000024
Figure JPOXMLDOC01-appb-T000024
Figure JPOXMLDOC01-appb-T000025
Figure JPOXMLDOC01-appb-T000025
Figure JPOXMLDOC01-appb-T000026
Figure JPOXMLDOC01-appb-T000026
　次に、前記１１２ＤＭＲセットから選んだ３か所のＤＭＲ全ての組み合わせに基づく２２７，９２０個のロジスティック回帰モデルを作成した。得られた判別式について、感度が９５％である判別式７９個を選択したところ、この中に出現するＤＭＲは５８か所であった（表中、５８ＤＭＲ）。さらに、判別式７９個に出現するＤＭＲの頻度を求めたところ、４回以上出現するものは１１か所であった（表中、１１ＤＭＲ）。 Next, 227,920 logistic regression models based on combinations of all three DMRs selected from the 112 DMR set were created. When 79 discriminants having a sensitivity of 95% were selected from the obtained discriminants, 58 DMRs appeared in the discriminant (58 DMR in the table). Furthermore, when the frequency of DMR appearing in 79 discriminants was determined, 11 were found 4 times or more (11 DMR in the table).
（２）ＤＭＲバイオマーカー候補を用いた臨床サンプルの多変量解析
　前記１１２ＤＭＲセットのメチル化率に基づき、実施例２の全４８サンプルのクラスター解析及び主成分分析を行った。この結果、クラスター解析では、大多数のＵＣ癌患者サンプルが同一クラスター（図１１中、枠内）に集積した。また、主成分分析（図１２）では、ＵＣ癌患者サンプル（●）と非癌ＵＣ患者サンプル（▲）が第１主成分方向にそれぞれ独立のクラスターを形成した。 (2) Multivariate analysis of clinical samples using DMR biomarker candidates Cluster analysis and principal component analysis of all 48 samples of Example 2 were performed based on the methylation rate of the 112 DMR set. As a result, in the cluster analysis, the majority of UC cancer patient samples accumulated in the same cluster (in the frame in FIG. 11). In the principal component analysis (FIG. 12), the UC cancer patient sample (●) and the non-cancer UC patient sample (▲) formed independent clusters in the first principal component direction.
（３）ＤＭＲバイオマーカー候補を用いて臨床サンプルの大腸癌発症可能性の評価
　前記１１２ＤＭＲセットのうち、ＤＭＲ番号２（ＳＩＸ１０）、１０（ＣＥＰ１１２）、５５（ＨＮＦ４Ａ）の領域のメチル化率をマーカーとした場合の、潰瘍性大腸炎患者の大腸癌発症の有無の判定の精度を調べた。 (3) Evaluation of likelihood of developing colon cancer in clinical samples using DMR biomarker candidates Markers of methylation rate in the region of DMR number 2 (SIX10), 10 (CEP112), 55 (HNF4A) in the 112 DMR set The accuracy of the determination of the presence or absence of colon cancer in patients with ulcerative colitis was investigated.
　具体的には、２４名のＵＣ癌患者及び２４名の非癌ＵＣ患者の直腸から採取された検体の前記３か所のＤＭＲのメチル化レベルの数値（β値）を用いてロジスティック回帰モデルに基づく判別式を作成してＵＣ癌患者と非癌ＵＣ患者の判別を行った。この結果、感度（ＵＣ癌患者のうち、陽性と評価された患者の割合）が９５．８％、特異度（非癌ＵＣ患者のうち、陰性と評価された患者の割合）が９５．８％、陽性的中率（陽性と評価された患者のうち、ＵＣ癌患者の割合）が９５．８％、陰性的中率（陰性と評価された患者のうち、非癌ＵＣ患者の割合）が９５．８％であり、いずれも９５％以上と高かった。また、図１３にＲＯＣ曲線を示す。この結果、ＡＵＣ（ＲＯＣ曲線下面積）は０．９７４であった。これらの結果から、前記１１２ＤＭＲセットから選択された数か所のＤＭＲの平均メチル化率に基づいて、高感度かつ高特異度で、潰瘍性大腸炎患者の大腸癌発症の可能性を評価できることが確認された。 Specifically, a logistic regression model was obtained using the numerical values (β values) of the three DMR methylation levels of specimens collected from the rectums of 24 UC cancer patients and 24 non-cancer UC patients. Based on the discriminant, a UC cancer patient and a non-cancer UC patient were discriminated. As a result, the sensitivity (the ratio of patients evaluated as positive among UC cancer patients) was 95.8%, and the specificity (the ratio of patients evaluated as negative among non-cancer UC patients) was 95.8%. The positive predictive value (the proportion of UC cancer patients out of those evaluated as positive) is 95.8%, and the negative predictive value (the proportion of non-cancer UC patients out of patients evaluated as negative) is 95%. .8%, both being as high as 95% or more. FIG. 13 shows the ROC curve. As a result, AUC (area under the ROC curve) was 0.974. From these results, it is possible to evaluate the possibility of developing colon cancer in patients with ulcerative colitis with high sensitivity and high specificity based on the average methylation rate of several DMRs selected from the 112 DMR set. confirmed.
１…大腸粘膜採取用キット、２、２Ａ、２Ｂ、２Ｃ…採取具、３ａ…第１の挟持片、３ｂ…第２の挟持片、４…連結部、５…挟持面、５ａ…第１の挟持面、５ｂ…第２の挟持面、６ａ、６ａ’…第１の挟持面５ａの辺縁部、６ｂ、６ｂ’…第２の挟持面５ｂの辺縁部、７ａ…第１の屈曲部、７ｂ…第２の屈曲部、８ａ、８ｂ…突起部、９ａ、９ｂ…筒部、１０ａ…緩衝部、１０ｂ…弾性部、１１、１１Ａ、１１Ｂ…採取補助具、１２…採取具導入部、１３…スリット、１４…把持部、１５…先端辺縁部、１６…手元辺縁部。 DESCRIPTION OF SYMBOLS 1 ... Colorectal mucosa sampling kit 2, 2A, 2B, 2C ... Collection tool, 3a ... 1st clamping piece, 3b ... 2nd clamping piece, 4 ... Connection part, 5 ... clamping surface, 5a ... 1st Clamping surface, 5b... 2nd clamping surface, 6a, 6a '.. edge portion of first clamping surface 5a, 6b, 6b' .. edge portion of second clamping surface 5b, 7a. 7b ... second bent portion, 8a, 8b ... projection portion, 9a, 9b ... cylindrical portion, 10a ... buffer portion, 10b ... elastic portion, 11, 11A, 11B ... collecting aid, 12 ... collecting device introduction portion, 13 ... slit, 14 ... gripping part, 15 ... tip edge part, 16 ... hand edge part.

Claims (34)


  1. 　ヒト潰瘍性大腸炎患者の大腸癌発症可能性を判定する方法であって、
    　ヒト潰瘍性大腸炎患者から採取された生体試料から回収されたＤＮＡ中の、表１～４に記載のメチル化可変領域番号１～１１２で表される各メチル化可変領域中に存在する１個以上のＣｐＧサイトのメチル化率を測定する測定工程と、
    　前記測定工程において測定されたメチル化率に基づいて算出されたメチル化可変領域の平均メチル化率と、予め設定された基準値又は予め設定された多変量判別式に基づいて、前記ヒト潰瘍性大腸炎患者の大腸癌発症の可能性を判定する判定工程
    を有し、
    　前記メチル化可変領域の平均メチル化率が、当該メチル化可変領域中のＣｐＧサイトのうち、前記測定工程においてメチル化率が測定された全てのＣｐＧサイトのメチル化率の平均値であり、
    　前記基準値が、各メチル化可変領域の平均メチル化率に対してそれぞれ設定された、発癌潰瘍性大腸炎患者と非癌潰瘍性大腸炎患者を識別するための値であり、
    　前記多変量判別式が、前記メチル化可変領域番号１～１１２で表されるメチル化可変領域のうちの１か所以上のメチル化可変領域の平均メチル化率を変数として含む、
    大腸癌発症可能性の判定方法。
    Figure JPOXMLDOC01-appb-T000001
    Figure JPOXMLDOC01-appb-T000002
    Figure JPOXMLDOC01-appb-T000003
    Figure JPOXMLDOC01-appb-T000004
    A method for determining the likelihood of developing colorectal cancer in a patient with human ulcerative colitis,
    One DNA present in each methylation variable region represented by methylation variable region numbers 1-112 shown in Tables 1 to 4 in DNA collected from a biological sample collected from a patient with human ulcerative colitis A measuring step for measuring the methylation rate of the above CpG site;
    Based on the average methylation rate of the methylation variable region calculated based on the methylation rate measured in the measurement step, and a preset reference value or a preset multivariate discriminant, the human ulcerative property A determination step of determining the likelihood of developing colon cancer in a patient with colitis,
    The average methylation rate of the methylation variable region is an average value of the methylation rates of all CpG sites whose methylation rates were measured in the measurement step among the CpG sites in the methylation variable region,
    The reference value is a value for discriminating between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient, each set for the average methylation rate of each methylation variable region,
    The multivariate discriminant includes, as a variable, an average methylation rate of one or more methylation variable regions among the methylation variable regions represented by the methylation variable region numbers 1-112.
    A method for determining the likelihood of developing colorectal cancer.
    Figure JPOXMLDOC01-appb-T000001
    Figure JPOXMLDOC01-appb-T000002
    Figure JPOXMLDOC01-appb-T000003
    Figure JPOXMLDOC01-appb-T000004

  2. 　前記判定工程において、メチル化可変領域番号１、３～２０、２３～２８、３１～４６、４９～６０、６２、６５～６９、７１、７３、７４、７９、８１、８２、８４、８６、８７、９０～９２、９５、１０１、１０３、１０９、１１０、及び１１２で表されるメチル化可変領域のうち１か所以上が、平均メチル化率が予め設定された基準値以下である、又は、メチル化可変領域番号２、２１、２２、２９、３０、４７、４８、６１，６３、６４、７０、７２、７５～７８、８０、８３、８５、８８、８９、９３、９４、９６～１００、１０２、１０４～１０８、及び１１１で表されるメチル化可変領域のうち１か所以上が、平均メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項１に記載の大腸癌発症可能性の判定方法。
    In the determination step, methylation variable region numbers 1, 3 to 20, 23 to 28, 31 to 46, 49 to 60, 62, 65 to 69, 71, 73, 74, 79, 81, 82, 84, 86, One or more of the methylation variable regions represented by 87, 90 to 92, 95, 101, 103, 109, 110, and 112 has an average methylation rate equal to or lower than a preset reference value; or , Methylation variable region numbers 2, 21, 22, 29, 30, 47, 48, 61, 63, 64, 70, 72, 75 to 78, 80, 83, 85, 88, 89, 93, 94, 96 to The human ulcerative colitis patient when one or more of the methylation variable regions represented by 100, 102, 104 to 108, and 111 has an average methylation rate equal to or higher than a preset reference value May have developed colorectal cancer It determines that the high, colon cancer onset possibility determination method according to claim 1.

  3. 　前記測定工程において、前記多変量判別式がその平均メチル化率を変数として含むメチル化可変領域中に存在する１個以上のＣｐＧサイトのメチル化率を測定し、
    　前記判定工程において、前記測定工程において測定されたメチル化率に基づいて算出されたメチル化可変領域の平均メチル化率と前記多変量判別式に基づいて当該多変量判別式の値である判別値を算出し、当該判別値が予め設定された基準判別値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項１に記載の大腸癌発症可能性の判定方法。
    In the measurement step, the multivariate discriminant measures the methylation rate of one or more CpG sites present in the methylation variable region including the average methylation rate as a variable,
    In the determination step, a discriminant value that is a value of the multivariate discriminant based on the average methylation rate of the methylation variable region calculated based on the methylation rate measured in the measurement step and the multivariate discriminant The human ulcerative colitis is determined to have a high possibility of developing colorectal cancer when the discriminant value is greater than or equal to a preset reference discriminant value. A method for determining the likelihood of developing colorectal cancer.

  4. 　前記多変量判別式が、メチル化可変領域番号１～１１２で表されるメチル化可変領域から選択される２か所以上のメチル化可変領域の平均メチル化率を変数として含む、請求項３に記載の大腸癌発症可能性の判定方法。
    4. The multivariate discriminant includes, as a variable, an average methylation rate of two or more methylation variable regions selected from methylation variable regions represented by methylation variable region numbers 1-112. The method for determining the likelihood of developing colorectal cancer as described.

  5. 　前記多変量判別式が、メチル化可変領域番号１～１１２で表されるメチル化可変領域から選択される３か所以上のメチル化可変領域の平均メチル化率を変数として含む、請求項３に記載の大腸癌発症可能性の判定方法。
    The multivariate discriminant includes, as a variable, an average methylation rate of three or more methylation variable regions selected from methylation variable regions represented by methylation variable region numbers 1-112 as variables. The method for determining the likelihood of developing colorectal cancer as described.

  6. 　前記多変量判別式が、メチル化可変領域番号１～５８で表されるメチル化可変領域からなる群より選択される１か所以上のメチル化可変領域の平均メチル化率を変数として含む、請求項３に記載の大腸癌発症可能性の判定方法。
    The multivariate discriminant includes, as a variable, an average methylation rate of one or more methylation variable regions selected from the group consisting of methylation variable regions represented by methylation variable region numbers 1 to 58. Item 4. A method for determining the likelihood of developing colorectal cancer according to Item 3.

  7. 　前記多変量判別式が、メチル化可変領域番号１～１１で表されるメチル化可変領域からなる群より選択される１か所以上のメチル化可変領域の平均メチル化率を変数として含む、請求項３に記載の大腸癌発症可能性の判定方法。
    The multivariate discriminant includes, as a variable, an average methylation rate of one or more methylation variable regions selected from the group consisting of methylation variable regions represented by methylation variable region numbers 1 to 11. Item 4. A method for determining the likelihood of developing colorectal cancer according to Item 3.

  8. 　ヒト潰瘍性大腸炎患者の大腸癌発症可能性を判定する方法であって、
    　ヒト潰瘍性大腸炎患者から採取された生体試料から回収されたＤＮＡ中の、配列番号１～８０で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を測定する測定工程と、
    　前記測定工程において測定されたメチル化率と、予め設定された基準値又は予め設定された多変量判別式に基づいて、前記ヒト潰瘍性大腸炎患者の大腸癌発症の可能性を判定する判定工程
    を有し、
    　前記基準値が、各ＣｐＧサイトのメチル化率に対してそれぞれ設定された、発癌潰瘍性大腸炎患者と非癌潰瘍性大腸炎患者を識別するための値であり、
    　前記多変量判別式が、前記配列番号１～８０で表される塩基配列中のＣｐＧサイトのうち１か所以上のＣｐＧサイトのメチル化率を変数として含む、
    大腸癌発症可能性の判定方法。
    A method for determining the likelihood of developing colorectal cancer in a patient with human ulcerative colitis,
    One or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 in DNA collected from biological samples collected from human ulcerative colitis patients A measurement process for measuring the methylation rate;
    Determination step of determining the possibility of developing colon cancer in the human ulcerative colitis patient based on the methylation rate measured in the measurement step and a preset reference value or a preset multivariate discriminant Have
    The reference value is a value for discriminating between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient, each set for the methylation rate of each CpG site,
    The multivariate discriminant includes, as a variable, the methylation rate of one or more CpG sites among the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80.
    A method for determining the likelihood of developing colorectal cancer.

  9. 　前記測定工程において、２～１０個のＣｐＧサイトのメチル化率を測定する、請求項８に記載の大腸癌発症可能性の判定方法。
    The method for determining the possibility of developing colorectal cancer according to claim 8, wherein the methylation rate of 2 to 10 CpG sites is measured in the measurement step.

  10. 　前記判定工程において、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、３１、４５、６４、６５、６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３～１０、１３、１９、２０、２５、２８、３０、３２～４４、４６～６３、６６、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８又は９に記載の大腸癌発症可能性の判定方法。
    In the determination step, the bases represented by SEQ ID NOs: 1, 2, 11, 12, 14-18, 21-24, 26, 27, 29, 31, 45, 64, 65, 67, 77, 79, and 80 One or more of the CpG sites in the sequence have a methylation rate equal to or lower than a preset reference value, or SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, 32 to 44 , 46-63, 66, 68-76, and 78, when one or more of the CpG sites in the base sequence have a methylation rate equal to or higher than a preset reference value, the human ulcer The method for determining the likelihood of developing colorectal cancer according to claim 8 or 9, wherein it is determined that the patient with ulcerative colitis is highly likely to develop colorectal cancer.

  11. 　前記測定工程において、配列番号１～３２で表される塩基配列中のＣｐＧサイトのメチル化率を測定し、
    　前記判定工程において、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、及び３１で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３～１０、１３、１９、２０、２５、２８、３０、及び３２で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８～１０のいずれか一項に記載の大腸癌発症可能性の判定方法。
    In the measurement step, the methylation rate of the CpG site in the base sequence represented by SEQ ID NOs: 1 to 32 is measured,
    In the determination step, one or more of the CpG sites in the base sequence represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, and 31 are methyl Of the CpG site in the base sequence represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32 The method according to any one of claims 8 to 10, wherein when the methylation rate is equal to or higher than a preset reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer. The method for determining the likelihood of developing colorectal cancer according to one item.

  12. 　前記判定工程において、配列番号１、２、１１、１２、１４～１８、２１～２４、２６、２７、２９、及び３１で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３～１０、１３、１９、２０、２５、２８、３０、及び３２で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が３以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８～１１のいずれか一項に記載の大腸癌発症可能性の判定方法。
    In the determination step, a methylation rate is set in advance among CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, and 31. The number of CpG sites that are equal to or less than the reference value and the methylation rate of CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32 are preliminarily determined. The human ulcerative colitis patient is determined to have a high possibility of developing colorectal cancer when the sum of the number of CpG sites equal to or greater than a set reference value is 3 or greater. The method for determining the likelihood of developing colorectal cancer according to any one of 11.

  13. 　前記測定工程において、配列番号１～１６で表される塩基配列中のＣｐＧサイトのメチル化率を測定し、
    　前記判定工程において、配列番号１、２、１１、１２、１４～１６で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３～１０、１３で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８～１０のいずれか一項に記載の大腸癌発症可能性の判定方法。
    In the measurement step, the methylation rate of the CpG site in the base sequence represented by SEQ ID NOs: 1 to 16 is measured,
    In the determination step, at least one of the CpG sites in the base sequence represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 16 has a methylation rate equal to or lower than a preset reference value. Alternatively, when one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10 and 13 have a methylation rate equal to or higher than a preset reference value, the human ulcerative colitis patient The method for determining the likelihood of developing colorectal cancer according to any one of claims 8 to 10, wherein it is determined that there is a high possibility that the patient has developed colorectal cancer.

  14. 　前記判定工程において、配列番号１、２、１１、１２、１４～１６で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３～１０、１３で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が３以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８～１０及び１３のいずれか一項に記載の大腸癌発症可能性の判定方法。
    In the determination step, among the CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 16, the number of CpG sites having a methylation rate equal to or lower than a preset reference value; The human ulcer when the sum of the CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10, 13 and the number of CpG sites having a methylation rate equal to or higher than a preset reference value is 3 or more The method for determining the likelihood of developing colorectal cancer according to any one of claims 8 to 10 and 13, wherein a patient with ulcerative colitis is determined to have a high probability of developing colorectal cancer.

  15. 　前記測定工程において、配列番号１～９で表される塩基配列中のＣｐＧサイトのメチル化率を測定し、
    　前記判定工程において、配列番号１及び２で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３～９で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８～１０のいずれか一項に記載の大腸癌発症可能性の判定方法。
    In the measurement step, the methylation rate of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 9 is measured,
    In the determination step, one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 1 and 2 have a methylation rate equal to or lower than a preset reference value, or SEQ ID NOs: 3 to 9 Possibility that the human ulcerative colitis patient develops colorectal cancer when one or more of the CpG sites in the represented base sequence has a methylation rate that is equal to or higher than a preset reference value The method for determining the likelihood of developing colorectal cancer according to any one of claims 8 to 10, wherein it is determined that the colorectal cancer is high.

  16. 　前記判定工程において、配列番号１及び２で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３～９で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が３以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８～１０及び１５のいずれか一項に記載の大腸癌発症可能性の判定方法。
    In the determination step, among the CpG sites in the base sequences represented by SEQ ID NOs: 1 and 2, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and SEQ ID NOs: 3 to 9 When the sum of the number of CpG sites having a methylation rate equal to or higher than a preset reference value among CpG sites in the base sequence is 3 or more, the human ulcerative colitis patient develops colorectal cancer The method for determining the likelihood of developing colorectal cancer according to any one of claims 8 to 10 and 15, wherein it is determined that there is a high possibility that the patient has a colorectal cancer.

  17. 　前記測定工程において、配列番号３３～６６で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を測定し、
    　前記判定工程において、配列番号４５、６４、及び６５で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３２～４４、４６～６３、及び６６で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８～１０のいずれか一項に記載の大腸癌発症可能性の判定方法。
    In the measurement step, the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66 is measured,
    In the determination step, at least one of the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65 has a methylation rate equal to or lower than a preset reference value, or SEQ ID NO: 32 A patient with human ulcerative colitis when one or more of the CpG sites in the base sequences represented by -44, 46-63, and 66 have a methylation rate equal to or higher than a preset reference value The method for determining the likelihood of developing colorectal cancer according to any one of claims 8 to 10, wherein it is determined that there is a high possibility that the patient has developed colorectal cancer.

  18. 　前記判定工程において、配列番号４５、６４、及び６５で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３２～４４、４６～６３、及び６６で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が２以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８～１０及び１７のいずれか一項に記載の大腸癌発症可能性の判定方法。
    In the determination step, among the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and SEQ ID NOs: 32-44 46 to 63 and 66, the sum of the number of CpG sites having a methylation rate equal to or higher than a preset reference value among CpG sites in the base sequences represented by 46 to 63 and 66 is 2 or more. The method for determining the likelihood of developing colorectal cancer according to any one of claims 8 to 10 and 17, wherein it is determined that a patient with ulcerative colitis is highly likely to develop colorectal cancer.

  19. 　前記測定工程において、配列番号３３、３５、３６、４３、６７～８０で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を測定し、
    　前記判定工程において、配列番号６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以下である、又は、配列番号３３、３５、３６、４３、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトのうち１か所以上が、メチル化率が予め設定された基準値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８～１０のいずれか一項に記載の大腸癌発症可能性の判定方法。
    In the measurement step, the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequence represented by SEQ ID NOs: 33, 35, 36, 43, 67-80 is measured,
    In the determination step, at least one of the CpG sites in the base sequences represented by SEQ ID NOs: 67, 77, 79, and 80 has a methylation rate equal to or lower than a preset reference value, or a sequence When one or more of the CpG sites in the base sequences represented by the numbers 33, 35, 36, 43, 68 to 76, and 78 have a methylation rate that is equal to or higher than a preset reference value, The method for determining the likelihood of developing colorectal cancer according to any one of claims 8 to 10, wherein it is determined that a human ulcerative colitis patient is likely to develop colorectal cancer.

  20. 　前記判定工程において、配列番号６７、７７、７９、及び８０で表される塩基配列中のＣｐＧサイトのうち、メチル化率が予め設定された基準値以下であるＣｐＧサイトの数と、配列番号３３、３５、３６、４３、６８～７６、及び７８で表される塩基配列中のＣｐＧサイトのうちメチル化率が予め設定された基準値以上であるＣｐＧサイトの数との和が２以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８～１０及び１９のいずれか一項に記載の大腸癌発症可能性の判定方法。
    In the determination step, among the CpG sites in the base sequences represented by SEQ ID NOs: 67, 77, 79, and 80, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and SEQ ID NO: 33 , 35, 36, 43, 68 to 76, and 78, the sum of the number of CpG sites having a methylation rate equal to or higher than a preset reference value among the CpG sites in the base sequence represented by 2 or more The method for determining the likelihood of developing colorectal cancer according to any one of claims 8 to 10 and 19, wherein the human ulcerative colitis patient is determined to have a high possibility of developing colorectal cancer.

  21. 　前記和が５以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項１２、１４、１６、１８、又は２０に記載の大腸癌発症可能性の判定方法。
    The colorectal cancer according to claim 12, 14, 16, 18, or 20, wherein when the sum is 5 or more, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer. How to determine the likelihood of onset.

  22. 　前記多変量判別式が、配列番号３３～６６で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を変数として含み、
    　前記測定工程において、前記多変量判別式がそのメチル化率を変数として含むＣｐＧサイトのメチル化率を測定し、
    　前記判定工程において、前記測定工程において測定されたメチル化率と前記多変量判別式に基づいて当該多変量判別式の値である判別値を算出し、当該判別値が予め設定された基準判別値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８又は９に記載の大腸癌発症可能性の判定方法。
    The multivariate discriminant includes, as a variable, the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66,
    In the measurement step, the multivariate discriminant measures the methylation rate of the CpG site including the methylation rate as a variable,
    In the determination step, a discriminant value that is a value of the multivariate discriminant is calculated based on the methylation rate measured in the measurement step and the multivariate discriminant, and the discriminant value is set in advance as a reference discriminant value. 10. The method for determining the likelihood of developing colorectal cancer according to claim 8 or 9, wherein, in the case described above, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.

  23. 　前記多変量判別式が、配列番号３３、３５、３６、４３、６７～８０で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトのメチル化率を変数として含み、
    　前記測定工程において、前記多変量判別式がそのメチル化率を変数として含むＣｐＧサイトのメチル化率を測定し、
    　前記判定工程において、前記測定工程において測定されたメチル化率と前記多変量判別式に基づいて当該多変量判別式の値である判別値を算出し、当該判別値が予め設定された基準判別値以上である場合に、前記ヒト潰瘍性大腸炎患者が大腸癌を発症している可能性が高いと判定する、請求項８又は９に記載の大腸癌発症可能性の判定方法。
    The multivariate discriminant is a variable of the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, 67-80. Including as
    In the measurement step, the multivariate discriminant measures the methylation rate of the CpG site including the methylation rate as a variable,
    In the determination step, a discriminant value that is a value of the multivariate discriminant is calculated based on the methylation rate measured in the measurement step and the multivariate discriminant, and the discriminant value is set in advance as a reference discriminant value. 10. The method for determining the likelihood of developing colorectal cancer according to claim 8 or 9, wherein, in the case described above, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.

  24. 　前記多変量判別式が、ロジスティック回帰式、線形判別式、ナイーブベイズ分類器で作成された式、又はサポートベクターマシンで作成された式である、請求項１～２３のいずれか一項に記載の大腸癌発症可能性の判定方法。
    The multivariate discriminant is a logistic regression equation, a linear discriminant, a formula created with a naive Bayes classifier, or a formula created with a support vector machine. A method for determining the likelihood of developing colorectal cancer.

  25. 　前記生体試料が、腸管組織である、請求項１～２４のいずれか一項に記載の大腸癌発症可能性の判定方法。
    The method for determining the likelihood of developing colon cancer according to any one of claims 1 to 24, wherein the biological sample is intestinal tissue.

  26. 　前記生体試料が、直腸粘膜組織である、請求項１～２５のいずれか一項に記載の大腸癌発症可能性の判定方法。
    The method for determining the likelihood of developing colorectal cancer according to any one of claims 1 to 25, wherein the biological sample is a rectal mucosa tissue.

  27. 　前記直腸粘膜組織が、
    　採取具と、採取補助具とを備え、
    　前記採取具は、
    　一方の端部に大腸粘膜を挟持する第１の挟持面が形成されている板状の第１の挟持片と、
    　一方の端部に大腸粘膜を挟持する第２の挟持面が形成されている板状の第２の挟持片と、
    　前記第１の挟持片と前記第２の挟持片を、互いに対向した状態で、前記第１の挟持面及び前記第２の挟持面が形成されていない端部において連結する連結部と、
    を有し、
    　前記第１の挟持面及び前記第２の挟持面の少なくとも一方がカップ形状であり、
    　前記採取補助具は、
    　側壁にスリットを有する円錐台形状の採取具導入部と、
    　棒状の把持部と、
    を有し、
    　前記把持部の一端が、前記採取具導入部の外径が大きい方の辺縁部近傍に連結しており、
    　前記スリットは、前記採取具導入部の外径が小さい方の辺縁部から外径が大きい方の辺縁部に向かって設けられており、
    　前記スリットの幅が、前記第１の挟持片の一方の端部と前記第２の挟持片の一方の端部の幅よりも広く、
    　前記採取具導入部の大きい方の外径が３０～７０ｍｍであり、回転軸方向の長さが５０～１５０ｍｍである、大腸粘膜採取用キットによって採取されたものである、請求項２６に記載の大腸癌発症可能性の判定方法。
    The rectal mucosa tissue is
    A collection tool and a collection aid;
    The sampling tool is
    A plate-like first clamping piece in which a first clamping surface for clamping the colonic mucosa is formed at one end;
    A plate-like second clamping piece in which a second clamping surface for clamping the colonic mucosa is formed at one end;
    A connecting portion that connects the first clamping piece and the second clamping piece in a state of being opposed to each other at an end portion where the first clamping surface and the second clamping surface are not formed;
    Have
    At least one of the first clamping surface and the second clamping surface is cup-shaped,
    The collection aid is
    A frustoconical sampling tool introduction part having a slit on the side wall;
    A rod-shaped gripping part;
    Have
    One end of the gripping part is connected to the vicinity of the edge part of the larger outer diameter of the sampling tool introduction part,
    The slit is provided from the edge of the smaller outer diameter of the sampling tool introduction part toward the edge of the larger outer diameter,
    The width of the slit is wider than the width of one end of the first clamping piece and the one end of the second clamping piece,
    27. The large-sized outer diameter of the collection tool introduction part is 30 to 70 mm, and the length in the rotation axis direction is 50 to 150 mm. A method for determining the likelihood of developing colorectal cancer.

  28. 　前記採取具が、
    　前記第１の挟持片の中心部よりも前記第１の挟持面が形成されている端部側に、第１の屈曲部を有し、
    　前記第２の挟持片の中心部よりも前記第２の挟持面が形成されている端部側に、第２の屈曲部を有する、請求項２７に記載の大腸癌発症可能性の判定方法。
    The sampling tool is
    Having a first bent portion on the end side where the first clamping surface is formed with respect to the center of the first clamping piece;
    28. The method for determining the likelihood of developing colorectal cancer according to claim 27, further comprising a second bent portion on an end portion side where the second clamping surface is formed with respect to a center portion of the second clamping piece.

  29. 　採取具と、採取補助具とを備える大腸粘膜採取用キットであり、
    　前記採取具は、
    　一方の端部に大腸粘膜を挟持する第１の挟持面が形成されている板状の第１の挟持片と、
    　一方の端部に大腸粘膜を挟持する第２の挟持面が形成されている板状の第２の挟持片と、
    　前記第１の挟持片と前記第２の挟持片を、互いに対向した状態で、前記第１の挟持面及び前記第２の挟持面が形成されていない端部において連結する連結部と、
    を有し、
    　前記第１の挟持面及び前記第２の挟持面の少なくとも一方がカップ形状であり、
    　前記採取補助具は、
    　側壁にスリットを有する円錐台形状の採取具導入部と、
    　棒状の把持部と、
    を有し、
    　前記把持部の一端が、前記採取具導入部の外径が大きい方の辺縁部近傍に連結しており、
    　前記スリットは、前記採取具導入部の外径が小さい方の辺縁部から外径が大きい方の辺縁部に向かって設けられており、
    　前記スリットの幅が、前記第１の挟持片の一方の端部と前記第２の挟持片の一方の端部の幅よりも広く、
    　前記採取具導入部の大きい方の外径が３０～７０ｍｍであり、回転軸方向の長さが５０～１５０ｍｍである、大腸粘膜採取用キット。
    A kit for collecting large intestine mucosa comprising a collection tool and a collection aid,
    The sampling tool is
    A plate-like first clamping piece in which a first clamping surface for clamping the colonic mucosa is formed at one end;
    A plate-like second clamping piece in which a second clamping surface for clamping the colonic mucosa is formed at one end;
    A connecting portion that connects the first clamping piece and the second clamping piece in a state of being opposed to each other at an end portion where the first clamping surface and the second clamping surface are not formed;
    Have
    At least one of the first clamping surface and the second clamping surface is cup-shaped,
    The collection aid is
    A frustoconical sampling tool introduction part having a slit on the side wall;
    A rod-shaped gripping part;
    Have
    One end of the gripping part is connected to the vicinity of the edge part of the larger outer diameter of the sampling tool introduction part,
    The slit is provided from the edge of the smaller outer diameter of the sampling tool introduction part toward the edge of the larger outer diameter,
    The width of the slit is wider than the width of one end of the first clamping piece and the one end of the second clamping piece,
    A large intestine mucosa collection kit having a larger outer diameter of the collection tool introduction part of 30 to 70 mm and a length in the rotation axis direction of 50 to 150 mm.

  30. 　前記採取具が、
    　前記第１の挟持片の中心部よりも前記第１の挟持面が形成されている端部側に、第１の屈曲部を有し、
    　前記第２の挟持片の中心部よりも前記第２の挟持面が形成されている端部側に、第２の屈曲部を有する、請求項２９に記載の大腸粘膜採取用キット。
    The sampling tool is
    Having a first bent portion on the end side where the first clamping surface is formed with respect to the center of the first clamping piece;
    30. The large intestine mucosa collection kit according to claim 29, further comprising a second bent portion on an end side where the second holding surface is formed with respect to a center portion of the second holding piece.

  31. 　前記第１の挟持面と前記第２の挟持面の両方がカップ形状である、請求項２９又は３０に記載の大腸粘膜採取用キット。
    The colonic mucosa collection kit according to claim 29 or 30, wherein both of the first clamping surface and the second clamping surface are cup-shaped.

  32. 　前記採取補助具が、回転軸方向に貫通孔を有し、前記採取具導入部の大きい方の外径が３０～７０ｍｍ、回転軸方向の長さが５０～１５０ｍｍであり、
    　前記カップ形状の辺縁部の内径が２～３ｍｍである、請求項２９～３１のいずれか一項に記載の大腸粘膜採取用キット。
    The collection aid has a through hole in the rotation axis direction, the outer diameter of the larger one of the collection tool introduction part is 30 to 70 mm, and the length in the rotation axis direction is 50 to 150 mm;
    The colonic mucosa collection kit according to any one of claims 29 to 31, wherein an inner diameter of the cup-shaped edge is 2 to 3 mm.

  33. 　前記第１の挟持面と前記第２の挟持面の辺縁部が鋸歯状である、請求項２９～３２のいずれか一項に記載の大腸粘膜採取用キット。
    The large intestine mucosa collection kit according to any one of claims 29 to 32, wherein edge portions of the first clamping surface and the second clamping surface are serrated.

  34. 　配列番号１～８０で表される塩基配列中のＣｐＧサイトからなる群より選択される１か所以上のＣｐＧサイトを含む部分塩基配列を有するＤＮＡ断片からなり、潰瘍性大腸炎患者の大腸癌発症可能性を判定するために用いられる、ＤＮＡメチル化率分析用マーカー。
    Onset of colorectal cancer in patients with ulcerative colitis, comprising a DNA fragment having a partial base sequence containing one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 DNA methylation rate analysis marker used to determine the possibility.
PCT/JP2017/024956 2016-07-08 2017-07-07 Method for judging onset possibility of large bowel cancer WO2018008740A1 (en)

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EP21152410.3A EP3845669A1 (en) 2016-07-08 2017-07-07 Method for judging onset possibility of large bowel cancer
CN201780041963.8A CN109415771A (en) 2016-07-08 2017-07-07 The determination method of colorectal cancer initiation potential
EP17824343.2A EP3483282A4 (en) 2016-07-08 2017-07-07 Method for judging onset possibility of large bowel cancer
EP21211939.0A EP4023162A2 (en) 2016-07-08 2017-07-07 Method for judging onset possibility of large bowel cancer
US16/315,961 US20190241970A1 (en) 2016-07-08 2017-07-07 Method for determining likelihood of colorectal cancer development
KR1020197000171A KR20190045146A (en) 2016-07-08 2017-07-07 How to determine the likelihood of developing colon cancer
JP2018526453A JP7094881B2 (en) 2016-07-08 2017-07-07 Method for determining the possibility of developing colorectal cancer
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